Sequence,Distance,andAccessibilityAreDeterminantsof -End ... · Quantitative Analysis of RNase H Cleavage Products—Band intensi- ties of cleavage products were determined using

Sequence, Distance, and Accessibility Are Determinants of5�-End-directed Cleavages by Retroviral RNases H*

Received for publication, September 26, 2005, and in revised form, November 11, 2005 Published, JBC Papers in Press, November 22, 2005, DOI 10.1074/jbc.M510504200

Sharon J. Schultz, Miaohua Zhang, and James J. Champoux1

From the Department of Microbiology, School of Medicine, University of Washington, Seattle, Washington 98195

The RNase H activity of reverse transcriptase is essential for ret-roviral replication. RNA5�-end-directed cleavages represent a formof RNase H activity that is carried out on RNA/DNA hybrids thatcontain a recessed RNA 5�-end. Previously, the distance from theRNA 5�-end has been considered the primary determinant for thelocation of these cleavages. Employingmodel hybrid substrates andthe HIV-1 andMoloneymurine leukemia virus reverse transcripta-ses, we demonstrate that cleavage sites correlate with specificsequences and that the distance from the RNA 5�-end determinesthe extent of cleavage. An alignment of sequences flankingmultipleRNA 5�-end-directed cleavage sites reveals that both enzymesstrongly prefer A or U at the �1 position and C or G at the �2position, and additionally for HIV-1, A is disfavored at the �4 posi-tion. For both enzymes, 5�-end-directed cleavages occurred whensites were positioned between the 13th and 20th nucleotides fromthe RNA 5�-end, a distance termed the cleavage window. In exam-ining the importanceof accessibility to theRNA5�-end, itwas foundthat the extent of 5�-end-directed cleavages observed in substratescontaining a free recessedRNA5�-endwasmost comparable to sub-strates with a gap of two or three bases between the upstream anddownstream RNAs. Together these finding demonstrate that theselection of 5�-end-directed cleavage sites by retroviral RNases Hresults from a combination of nucleotide sequence, permissible dis-tance, and accessibility to the RNA 5�-end.

In reverse transcription, the single-stranded RNA genome of a retro-virus is transformed into double-stranded DNA by the viral-encodedreverse transcriptase (reviewed in Refs. 1 and 2). This multifunctionalenzyme carries out DNA synthesis, strand displacement synthesis, andstrand transfer and degrades the RNA portion of RNA/DNA hybrids.The polymerase domain of reverse transcriptase represents the amino-terminal two-thirds of the protein, whereas the RNase H domain com-prises the remaining carboxyl-terminal portion. Although the polymer-ase and RNase H activities are functionally separable, both activities areessential for retroviral replication. The reverse transcriptase of humanimmunodeficiency virus type 1 (HIV-1)2 functions as an asymmetricheterodimer composed of p66 and p51 subunits (3), whereas that ofMoloney murine leukemia virus (M-MuLV) is a 76-kDa protein thatmay function as a homodimer or as a monomer (4–6).RNase H contributes to reverse transcription in three distinct ways

(reviewed in Refs. 1, 7, and 8). First, RNase H carries out degradation of

the RNA genome both during and after minus-strandDNA synthesis tofacilitate plus-strand DNA synthesis and strand transfers. Second,RNase H creates the polypurine tract (PPT) primer from the viralgenome. And third, RNase H removes the PPT and tRNA primers usedto prime plus-strand and minus-stand DNA synthesis, respectively.Given the vital roles of RNase H in retroviral replication, it is importantto understand how RNase H recognizes the RNA/DNA hybrids createdduring reverse transcription as substrates for cleavage. Extensive studieshave shown that both generation of the PPT primer and removal of thePPT and tRNA primers require specific sequences (9–19). By contrast,fewer studies have examined general degradation by RNase H (20–23),and less is known about the determinants thatmight influence RNaseHspecificity for this type of degradation during retroviral replication.RNase H has both polymerization-dependent and polymerization-

independent modes of cleavage. The polymerization-dependent modeaccompanies RNA-dependent DNA synthesis, and the nascent DNA 3�primer terminus positions RNase H cleavages. Such cleavages occur17–20 nucleotides away on the RNA template strand but are not strictlycoupled to DNA synthesis (20, 24–26). This mode of cleavage likelyfunctions at pause sites during minus-strand synthesis, when the 3�terminus of the nascent DNA is recessed on the genomic template.However, the polymerization rate of reverse transcriptase is faster thanthe rate of RNase H cleavage (27), and the polymerization-dependentmode of RNase H cleavage does not completely degrade the templateRNA (20, 28). Thus significant amounts of RNA can remain annealed tothe minus-strand DNA (22), and removal of these fragments by thepolymerization-independent RNase H activity is likely necessary forefficient plus-strand DNA synthesis.Polymerization-independent RNase H cleavages have been attrib-

uted to at least three distinct mechanisms, which differ in how reversetranscriptase associates with the hybrid substrate (reviewed in Refs. 1, 7,and 8). First, reverse transcriptase can bind the RNA strand of a hybridwithout positioning by either aDNA3�-end or anRNA5�-end, resultingin internal cleavages by the RNase H (23). Second, the polymerasedomain can bind a recessed DNA 3� primer terminus to facilitate DNA3�-end-directed cleavages that occur �17–20 nucleotides back on theRNA strand of a hybrid, similar to what happens at pause sites duringpolymerization. Third, the polymerase domain can associate with arecessed RNA5�-end, andRNaseH can carry out 5�-end-directed cleav-ages that occur �15–20 nucleotides downstream on the RNA strand.While the distance from the recessed RNA 5�-end has been character-ized as the primary determinant for this mechanism (29), these cleav-ages have been observed as close as 12–15 nucleotides (30, 31) and as faras 21 nucleotides (29, 32) from the RNA 5�-end. Notably, this range ofdistances is much broader than themore fixed distance of 17–19 nucle-otides that separates the polymerase and RNase H active sites as deter-mined by crystallography studies or footprinting of reverse tran-scriptase with substrates (33–36).A limited number of studies has suggested the possibility that

sequencemight influence 5�-end-directed cleavages (24, 29, 30), but this

* This work was supported by National Institutes of Health Grant CA51605. The costs ofpublication of this article were defrayed in part by the payment of page charges. Thisarticle must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.Section 1734 solely to indicate this fact.

1 To whom correspondence should be addressed: Dept. of Microbiology, Box 357242,School of Medicine, University of Washington, Seattle, WA 98195-7242. Tel.: 206-543-8574; Fax: 206-543-8297; E-mail: [email protected].

2 The abbreviations used are: HIV-1, human immunodeficiency virus, type 1; M-MuLV,Moloney murine leukemia virus; PPT, polypurine tract; ribonuclease H, RNase H.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 4, pp. 1943–1955, January 27, 2006© 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

JANUARY 27, 2006 • VOLUME 281 • NUMBER 4 JOURNAL OF BIOLOGICAL CHEMISTRY 1943

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hypothesis has not been directly tested before. We recently reportedthat sequence specificity is an important determinant of internal RNaseH cleavages and that an RNA 5�-end at a nick does not promote 5�-end-directed RNaseH cleavages (23). These results prompted us to ask threefundamental questions concerning RNA 5�-end-directed cleavages byretroviral RNases H. First, does sequence influence 5�-end-directedcleavages? Second, what distances from the RNA 5�-end are acceptablefor 5�-end-directed cleavage sites? Third, how large of a gap is requiredbetween the 3� terminus of an upstreamRNA and the 5�-end of a down-stream RNA to allow efficient 5�-end-directed cleavages in the down-stream RNA? Our findings offer new insights into the mechanisms ofRNA 5�-end-directed cleavages for M-MuLV and HIV-1 reverse tran-scriptases, and the role of RNase H in retroviral replication.

EXPERIMENTAL PROCEDURES

Enzymes and Reagents—Recombinant HIV-1 reverse transcriptasewas obtained fromWorthington Biochemicals. Recombinant wild-typeM-MuLV reverse transcriptase, T7DNApolymerase, and calf intestinalalkaline phosphatase were purchased from Amersham Biosciences. T4polynucleotide kinase, T4 DNA ligase, T4 DNA polymerase, and allrestriction enzymes were obtained from New England Biolabs. DNAoligonucleotides were obtained from Qiagen and Invitrogen. Oligonu-cleotides were gel-purified in denaturing polyacrylamide gels asdescribed (23, 37).

Preparation of RNAs—Preparation of RNAs Md1-Md10, PPT20,PPT62, and NPPT has been described previously (23). To prepare R46RNA, a 46-mer DNA oligonucleotide (5�-CAATGAAAGACCCCAC-CTGTAGGTTTGGCAAGCTAGCTTCGAGACG-3�) was annealedto a 54-merDNAoligonucleotide (5�-AATTCGTCTCGAAGCTAGC-TTGCCAAACCTACAGGTGGGGTCTTTCATTGAGCT-3�), andthis duplex was introduced into EcoRI- and SacI-linearized pGEM9Z-f(�) (Promega). The resulting plasmid was linearized with BsmBI andtranscribed in vitro as described (23). All other RNAs were prepared byin vitro transcription of linearized plasmids as follows. Tomake 35-merR7Z1, pGEM7Zf(�) (Promega) was linearized with XbaI. To make 28-mer R9Z1, pGEM9Zf(�) was linearized with XbaI. To make 32-merRpKS1, pBluescript KSII(�) (Stratagene) was linearized with NotI. Tomake 30-mer R3Z1, pGEM3Zf(�) (Promega) was linearized withBamHI. Because RNA R3Z1 is identical in sequence to the 5�-end of a41-nucleotide RNA that has been used extensively to map 5�-end-dire-cted cleavages (29, 38–43), we have used the cleavage sites for RNAR3Z1 as mapped in our own experiments.To generate additional RNAs with different sequences, some pGEM

plasmids were digested with single-cutting restriction enzymes down-stream of the T7 promoter (as indicated below), the overhangs werefilled in or removed by T4 DNA polymerase treatment, the blunt endswere ligated using T4 DNA ligase, and the resulting plasmids weretransformed into XL1-Blue cells (Stratagene). The sequences of theseplasmids were confirmed byDNA sequencing. For T4DNApolymerasetreatment, a digested plasmid was incubated in a 20-�l reaction con-taining 1 mM dNTPs and 3 units of T4 DNA polymerase for 5 min at37 °C and then for 15 min at 12 °C. To make 25-mer R3Z2,pGEM3Zf(�) was digested by EcoRI and BamHI, and the resulting plas-mid was linearized with HindIII prior to in vitro transcription. Tomake27-mer R7Z2, pGEM7Zf(�) was digested by ApaI, treated with T4DNA polymerase, ligated, digested with AatII, treated with T4 DNApolymerase, and ligated, and then the resulting plasmid was linearizedwith XbaI. To make 32-mer R7Z3, pGEM7Zf(�) was digested by ApaIand XbaI, and the resulting plasmid was linearized with Acc65I. Tomake 30-mer R7Z4, pGEM7Zf(�) was digested by ApaI and EcoRI, and

the resulting plasmid was linearized with BstBI. To make 30-mer R7Z5,pGEM7Zf(�) was digested byApaI and SmaI, and the resulting plasmidwas linearized with HindIII.

5�-End Labeling, 3�-End Labeling, and 5�-End Phosphorylation—5�-End labeling or 3�-end labeling of RNAswere carried out as describedpreviously (23, 37). Because cleavage by RNase H produces a 3�-hy-droxyl group and a 5�-phosphate group, an unlabeled phosphate groupwas added to the 5�-ends of 3�-end-labeled RNAs as previouslydescribed (23).

Preparation of Hybrid Substrates—To prepare the substrate withoutupstream RNA (No Upstream substrate), RNAs were annealed to theappropriate template DNAs at an RNA:DNA molar ratio of 1:2, and toprepare the Nick or Gap substrates, upstream PPT20 RNA was addi-tionally included at a 3-foldmolar excess over the first RNA.Annealingswere carried out in 10 mM Tris-HCl, pH 8.0, and 200 mM KCl at 90 °Cfor 3 min followed by cooling to room temperature.For the No Upstream substrates, the RNA 5�-end was recessed by 22

nucleotides from the DNA 3�-end and the RNA 3�-end extended twobases beyond the DNA 5�-end. Hybrids containing RNAs Md1, Md2,Md4, Md6, Md7, Md9, and Md10 were annealed to DNA strands D49,D49�1, D49�3, D49�5, D49�6, D49�8, and D49�9, respectively (23).Hybrids containing RNAs R3Z1, R3Z2, R7Z1, R7Z2, R7Z3, R7Z4, R7Z5,R9Z1, R46, and RpKS2 were similarly positioned on DNA strands con-taining the appropriate complementary sequences. RNA NPPT wasannealed to D52N (23), and PPT62 was annealed to D�27temp4 (23).For the Nick substrate containing upstream PPT20 and downstream

Md1, the template strand was D49 (5�-CCAAACCTACAGGTGGGG-TCTTTCATTT[ ]CCCCCCTTTTTCTGGAGACTAA-3�). The brac-kets in the sequence of D49 indicate the position that nucleotides wereadded to create the template strands for the corresponding Gap subst-rates, which are D49(1b) (5� . . . [T] . . . 3�), D49(2b) (5� . . . [GT] . . . 3�),D49(3b) (5� . . . [AGT] . . . 3�), D49(4b) (5� . . . [CAGT] . . . 3�), andD49(5b) (5� . . . [ACAGT] . . . 3�).For the Nick substrate containing upstream PPT20 and downstream

Md10, the template strand was D49�9 (5�-GCTAGCTTGCCAAAC-CTACAGGTGGGG[ ]CCCCCCTTTTTCTGGAGACTAA-3�). Thebrackets in the sequence ofD49�9 indicate the position that nucleotideswere added to create the template strands for the corresponding Gapsubstrates, which are D49�9(1b) (5� . . . [T] . . . 3�), D49�9(2b) (5� . . .[GT] . . . 3�), D49�9(3b) (5� . . . [AGT] . . . 3�), D49�9(4b) (5� . . .[CAGT] . . . 3�), and D49�9(5b) (5� . . . [ACAGT] . . . 3�).

Cleavage Analysis of Hybrid Substrates—Three units (0.6–0.8 pmol)of M-MuLV reverse transcriptase or 0.5 unit (0.2 pmol) of HIV-1reverse transcriptase were incubatedwith 0.2 pmol (10 nM final concen-tration) of a hybrid substrate in 20-�l reactions containing 50 mM Tris-HCl, pH 8.0, 50 mM KCl, 6 mMMgCl2, and 5 mM dithiothreitol at 37 °Cfor the times indicated. Aliquots were added to formamide stop mix(95% formamide, 20mMEDTA) and electrophoresed in denaturing 20%polyacrylamide gels (National Diagnostics). Products were visualized byPhosphorImager analysis (Amersham Biosciences).

Statistical Analysis—For each position of the aligned sequences, thechi-square one-dimensional test was used to determine the deviationfrom the random distribution of bases (44). The expected frequency foreach basewas determined by summing the nucleotide frequencies of thefirst 25 nucleotides from the 5�-end of each RNA used for HIV-1 (Fig. 5)and for M-MuLV (Fig. 6). For HIV-1, the expected frequencies wereA � 0.233, C � 0.243, G � 0.319, and U � 0.205. For M-MuLV, theexpected frequencies were A � 0.220, C � 0.251, G � 0.326, and U �

0.203.

RNA 5�-End-directed Cleavages by RNases H

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Quantitative Analysis of RNase H Cleavage Products—Band intensi-ties of cleavage products were determined using ImageQuanT (Amer-sham Biosciences). To compare the amount of product resulting fromcleavage at the same sequence in different RNAs (for example, countingfrom the RNA 5�-end, site F is cleaved in Md1 between the 16th and17th nucleotides, and in Md2 between the 15th and 16th nucleotides),the area integration function was used to quantitate the relative abun-dance of individual bands present in each sample. This analysis normal-ized the amount of cleavage product for a specific site as a percentage ofthe total cleavage products for each hybrid substrate. Quantitationsusing data from the 15-s and 1-min time points were found to be essen-tially identical, and the results from 1-min time points are presentedgraphically in Fig. 4.

RESULTS

Sequence and Distance Are Determinants of RNA 5�-End-directedCleavages—The 5�-end-directed cleavages by RNase H have beenshown to occur 15–20 nucleotides from the RNA 5�-end. To addresshow RNase H cleaves at specific sites within this range, we have evalu-ated the roles of RNA sequence, distance, and accessibility to the RNA5�-end as possible determinants of cleavage. As a model system, hybridsubstrates with RNAs containing sequence from the M-MuLV PPTregion were used. In this region of the M-MuLV genome, the cleavagethat generates the 3�-end of the PPT primer is defined to occur betweennucleotides �1 and �1 and is called the �1/�1 cleavage site. Thisnumbering is also used to designate the positions of nucleotides in themodel hybrid substrates, which contained 7 different 29-mer RNAs,termed Md1 through Md10 (Fig. 1). These RNAs have 5�-ends begin-ning downstream of the PPT at positions �1, �2, �4, �6, �7, �9, or�10 and contain a total of 29 consecutive nucleotides of sequence, suchthat blocks of the same sequence are located at different distances fromthe RNA 5�-ends (shaded box in Fig. 1). These RNAs were used to ask if5�-end-directed cleavages occur at identical distances from the RNA5�-ends and if sequence influences the positioning of 5�-end-directedcleavages.For RNaseH cleavage assays, eachRNAwas 5�- or 3�-end-labeled and

annealed to a template DNA to generate a hybrid with a recessed RNA5�-end. Hybrid substrates were incubated with HIV-1 or M-MuLVreverse transcriptase in time-course assays; the products representingthe initial 5�-end-directed cleavage events were found in the 15-s and1-min time points when most of the initial substrate remaineduncleaved. These initial cleavage sites were mapped by comparing the

mobilities of cleavage products to size ladders generated with nucleaseP1 (data not shown). Depending upon whether an RNA was 5�- or3�-end-labeled, the longest products represented the cleavages thatoccur the furthest from or the closest to the RNA 5�-end, respectively.Importantly, the location and extent of cleavage for each sitewere deter-mined by using the data obtained for both 5�- and 3�-end-labeled RNAs.To facilitate comparison of the same sites in different substrates, thecleavage sites generating the observed products have been named Athrough I, as indicated in the relevant figures and as described previ-ously (23).When substrates containing 5�-end-labeled RNAs Md1 through

Md10were treatedwithHIV-1 reverse transcriptase, it was immediatelyapparent that the cleavage products were not of uniform size (Fig. 2A).Instead, the products had varied lengths indicating that cleavagesoccurred at different distances from the RNA 5�-ends. The expectedproducts resulting from cleavage at the same sites in the different sub-strates (labeled “E–I”) are connected by lines in Fig. 2. Substrate Md1was predominantly cleaved at sites E, F, and G (Fig. 2A, lanes 1–5).Counting from the RNA 5�-end, site E is between nucleotides 13 and 14,site F is between nucleotides 16 and 17, and site G, which representedthe most distal 5�-end-directed cleavage site in Md1, falls betweennucleotides 19 and 20. Cleavage of substrate Md2 was very similar tothat of substrate Md1 except that all of the cleavage products were onenucleotide smaller (lanes 6–10). Importantly, site G remained the mostdistal 5�-end-directed cleavage, even though this site was now locatedbetween the 18th and 19th nucleotides from theMd2 RNA 5�-end, and,unlike Md1, no cleavage occurred between the 19th and 20th nucleo-tides. Although site H was not cleaved in Md1 or Md2, this site wasdetectably cleaved between the 19th and 20th nucleotides in substrateMd4 (lanes 11–15) and significantly cleaved between the 17th and 18thnucleotides in substrate Md6 (lanes 16–20). No cleavage was observedat site I in substrates Md1 through Md7 (lanes 1–25), but this site wascleaved when located between the 19th and 20th nucleotides in sub-strate Md9 and between the 18th and 19th nucleotides in substrateMd10 (lanes 25–35).Assays with 3�-end-labeled RNAs and HIV-1 reverse transcriptase

were used to confirm that the 5�-end-directed cleavages sites closestto the RNA 5�-ends in the above analysis represented the initialcleavage products and were not the result of secondary cleavages(Fig. 2B). In general, the locations were the same as for the 5�-end-labeled substrates, but the relative abundance of products sometimesvaried because strong cleavage sites close to the labeled end dimin-

FIGURE 1. RNA oligonucleotides used in RNase Hcleavage assays. All RNAs contain M-MuLVsequences from the PPT region, which containsthe �1/�1 site where cleavage occurs to generatethe plus-strand primer and which corresponds tonucleotides 7815 and 7816 in the M-MuLVsequence (55). PPT20 is upstream of this site andspans nucleotides �20 to �1. The seven down-stream RNAs are named as Moloney downstream(Md) followed by the nucleotide position of the 5�terminus relative to the �1/�1 site. The sequenceshared by all of these RNAs is indicated by a shadedbox.




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ished the detection of more distal sites. Site E was cleaved well insubstrate Md1 at the earliest time points but was only weakly cleavedin subsequent substrates (lanes 1–20). Site F was a strong cleavagesite when located as close as between the 13th and 14th nucleotidesfrom the RNA 5�-end (Md1–Md4, lanes 1–15) but was not cleavedefficiently when located closer than between the 11th and 12thnucleotides (Md6–Md10, lanes 15–35). Site G became the strongestcleavage site close to the RNA 5�-end when located between the 14thand 15th nucleotides in substrate Md6 (lanes 16–20) and between

the 13th and 14th nucleotides in substrate Md7 (lanes 21–25). How-ever, this site was not cleaved when moved two bases closer to theRNA 5�-end in substrate Md9 (lanes 26–30). In substrates Md9 andMd10, the cleavage site recognized closest to the RNA 5�-end wassite H (lanes 26–30). Site I was not cleaved until located between the19th and 20th nucleotides inMd9 or the 18th and 19th nucleotides inMd10 (lanes 26–35).These same substrates were also used to analyze 5�-end-directed

cleavage by the RNase H of M-MuLV reverse transcriptase (Fig. 3).

FIGURE 2. Analysis of 5�-end-directed RNase Hcleavages in RNAs Md1 through Md10 usingHIV-1 reverse transcriptase. The hybrid sub-strate (thick line RNA) shown schematically at thetop of each panel contained the indicated 5�-end-labeled (A) or 3�-end-labeled (B) RNA. Substrateswere treated with HIV-1 reverse transcriptase, ali-quots were removed at the indicated times, andsamples were analyzed in denaturing 20% poly-acrylamide gels. Results were visualized using aPhosphorImager. Products resulting from cleav-age at specific sites in Md1 are labeled as sites E–I,and solid lines track cleavage products corre-sponding to products from cleavage of the samesites in the other substrates. In Md1 (see Fig. 1), thecleavage sites are found between the followingnucleotides from the RNA 5�-end: site E is �13/�14, site F is �16/�17, site G is �19/�20, site H is�22/�23, and site I is �27/�28. The cleavagesites identified in Md1 that give rise to the cleav-age products shown for Md10 are indicated at theright.




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Most of the cleavage sites recognized in Md1 through Md10 byM-MuLV reverse transcriptase were identical to those recognized bythe HIV-1 enzyme, but the extent of cleavage often varied. M-MuLVRNase H cleaved 5�-end-labeledMd1 at site D, between the 8th and 9thnucleotides, and at sites E and F, but notably, no 5�-end-directed cleav-ages occurred beyond site F (Fig. 3A, lanes 1–5). Faint cleavage of site Gwas apparent between the 18th and 19th nucleotides in substrate Md2but was more distinct in substrate Md4, when this site was locatedbetween the 16th and 17th nucleotides (lanes 6–15). Adjacent cleavagesbeginning at site H were the most distal 5�-end-directed cleavages insubstrates Md6 (between the 17th and 18th nucleotides) and Md7(between the 16th and 17th nucleotides) (lanes 16–25). Similarly insubstratesMd9 andMd10, a pair of cleavages between the 19th and 20thnucleotides or the 18th and 19th nucleotides, respectively, were thefurthest 5�-end-directed cleavage sites and included site I (lanes 26–30).

As described above, we used 3�-end-labeled RNAs in the hybrid sub-strates to map the 5�-end-directed cleavages close to the RNA 5�-endsforM-MuLV. In general, the strongest cleavages were observed at posi-tions corresponding to site F in substratesMd1,Md2, andMd4 (Fig. 3B,lanes 1–15). In substrates Md6 through Md10, adjacent cleavages,including site H, were the strongest cleavages near the RNA 5�-end(lanes 16–35). The locations of these cleavages ranged from betweenthe 17th and 18th nucleotides in Md6 to between the 13th and 14thnucleotides in Md10.The short fragment resulting from cleavage between the 8th and 9th

nucleotides at site D observed with the 5�-end-labeled substrate (Fig.3A, lane 2) could have been a secondary cleavage product. However, atleast some of this product was generated independent of other cleav-ages, because the corresponding cleavage product was also observedusing the 3�-end-labeled substrateMd1 (Fig. 3B, lanes 1–5). Limited but

FIGURE 3. Analysis of 5�-end-directed RNase Hcleavages in RNAs Md1 through Md10 usingM-MuLV reverse transcriptase. The hybrid sub-strate shown schematically at the top of each panelcontained the indicated 5�-end-labeled (A) or3�-end-labeled (B) RNA. Substrates were treatedwith M-MuLV reverse transcriptase and analyzedas described in Fig. 2. In Md1, the cleavage sites arefound between the following nucleotides fromthe RNA 5�-end: site D is �8/�9, site E is �13/�14,site F is �16/�17, site G is �19/�20, site H is �22/�23, and site I is �27/�28.




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detectable independent cleavages also occurred between the 8th and 9thnucleotides of two other substrates, at site E in substrateMd6 and at siteF in substrate Md9 (lanes 16–20 and 26–30, respectively). The rele-vance of these 5�-proximal cleavages to 5�-end-directed cleavage is con-sidered under “Discussion.”

Quantitative Analysis of Distance Effects on Extent of Cleavage—Tobetter understand how the extent of cleavage at a specific site is affectedby the distance of the site relative to the RNA 5�-end, we carried outquantitative analyses of the cleavage products generated with the5�-end-labeled substrates Md1 through Md10 shown in Figs. 2 and 3.Sites F, G, andHwere chosen as representative cleavages, because thesesites were recognized by both HIV-1 andM-MuLV reverse transcripta-ses and because they are present over the relevant range of distances inthe various substrates.First, the extent of cleavage at each site was evaluated in all substrates.

For HIV-1 RNase H, cleavage at site F appeared to have a bell-shapedpattern, with the highest cleavage occurring with substrate Md4 (Fig.4A). The pattern was similar for site G, but cleavage wasmost abundantin substrates Md6 and Md7. The pattern for cleavage of site H was alsosimilar, with the highest cleavage in substrates Md9 and Md10. ForM-MuLVRNaseH, the amount of cleavage at each site also appeared asnon-overlapping, bell-shaped patterns distributed in substrates Md1through Md10 (Fig. 4B). Cleavage of site F was maximal in substrateMd2, cleavage of site G was highest in substrates Md4 and Md6, and

cleavage of siteHwas greatest in substratesMd6 andMd7. Interestingly,the greatest amount of cleavage for a particular site occurred in differentsubstrates for M-MuLV and HIV-1 RNases H. For example, site F wascleaved to the greatest extent in substrate Md2 for M-MuLV RNase Hand in substrate Md4 for HIV-1 RNase H (Fig. 4AB).Next, we determined how the extent of cleavage at sites F, G, and H

was affected by the distance from the RNA 5�-end. For HIV-1 RNase H(Fig. 4C), the plots of distance versus cleavage at each site overlapped,indicating that the overall effects of distance on the extent of cleavagewere very similar. A plot of the equivalent data for M-MuLV RNase Halso showed that distance from the RNA 5�-end similarly influenced theextent of cleavage at sites F, G, and H (Fig. 4D).

Preferred Nucleotides Flank RNA 5�-End-directed Cleavage Sites—We next asked if preferred nucleotides could be identified near 5�-end-directed cleavage sites, because we recently showed that preferrednucleotides are found near internal cleavage sites recognized by theM-MuLVandHIV-1RNasesH (23). This analysis required themappingof 5�-end-directed cleavage sites on a variety of different hybrid sub-strates containing RNAs with recessed 5�-ends. Three RNAs derivedfrom viral sequences were used: PPT62, containing sequence from theM-MuLV genome (PPT62 (23)), and RNAs Md1 and Md10. To gener-ate additional sequence diversity, several RNAs with unique sequencesweremade using in vitro transcription plasmids (see “Experimental Pro-cedures”). Finally, for HIV-1 reverse transcriptase, we have included

FIGURE 4. Extent of cleavage and optimal distances for cleavage at sites F, G, and H in RNAs Md1 through Md10 by HIV-1 and M-MuLV reverse transcriptases. The amountof product generated by cleavage (% of total) at sites F, G, and H in the indicated substrates was determined for HIV-1 (A) or M-MuLV (B) reverse transcriptase. Data from the 1-min timepoints in three (A) or four (B) independent experiments with 5�-end-labeled RNAs were used to determine the amount of product that resulted from the cleavages at site F (gray bars),site G (black bars), or site H (white bars) (�S.D.). These same data were also used to analyze the optimal distance for cleavage of each site relative to the 5�-RNA ends for HIV-1 (C) orM-MuLV (D) reverse transcriptase. The amount of product generated by cleavage (% of total) for sites F (gray squares), G (black circles), or H (open triangles) is plotted as a function ofthe cleavage site distance in nucleotides from the 5�-end of each substrate.




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5�-end-directed cleavage sites reported previously by other investiga-tors in studies where the exact positions of these sites were mapped (21,25, 29, 38–43, 45).To generate hybrid substrates with recessed 5�-RNA ends, RNAs

were 5�- or 3�-end-labeled and annealed to DNA templates. These sub-strates were used in RNase H cleavage assays as shown above withHIV-1 or M-MuLV reverse transcriptase, and cleavage sites weremapped to the nucleotide level (data not shown). The relative extent ofcleavage at the various sites in each substrate was classified as strong,medium, or weak. As an example using substrate containing RNAMd1and HIV-1 reverse transcriptase (Fig. 2, A and B, lanes 1–5), sites E andF were classified as strong, and site G was classified as medium. Usingsubstrate Md1 with M-MuLV reverse transcriptase (Fig. 3, A and B,lanes 1–5), site E was classified as medium and site F was classified asstrong. For both M-MuLV and HIV-1, cleavages between sites E and Frepresent weak sites. In our analysis, only strong andmedium sites wereconsidered. For themapped cleavage sites observed in prior studieswithHIV-1 (21, 25, 29, 38–43, 45), all of the identified RNaseH cleavage siteswere used.To compare the sequences surrounding RNA 5�-end-directed cleav-

age sites, the cleavage was defined to occur between nucleotides�1 and�1, and the flanking sequences were aligned. These cleavage sites arepresented for HIV-1 in Fig. 5 and for M-MuLV in Fig. 6. To statisticallydetermine whether any base preferences correlated with the cleavagesites, the nucleotides from positions �10 to �4 were tabulated. Thefrequency of bases in the first 25 nucleotides of sequence beginningfrom each RNA 5�-end was calculated to determine the expected distri-bution of nucleotides. For a given cleavage site, the significance of anydeviations from random nucleotide frequencies was determined bycomparing the base distribution at each position with the expected dis-tribution using the chi-square method (“Experimental Procedures”).The resulting chi-square values were plotted against the nucleotidepositions (Fig. 7).For both HIV-1 andM-MuLV, two nucleotide positions had p values

�0.01 (�2 values 11.34) and were considered strong deviations fromrandom. Position �1 showed a strong preference for A or U, toleratedC, and strongly disfavored G. Position�2 had a strong preference for Gor C and especially disfavored U. In addition, the�4 position for HIV-1also had a p value �0.01 and disfavored A. Unlike the nucleotide pref-erences seen for internal cleavage sites (23), strong preferences forsequences further upstream were not observed (see “Discussion”).

Accessibility to a 5�-EndAffects RNA 5�-End-directed Cleavages—Werecently showed that the 5�-end of an RNA at a nick does not allow5�-end-directed cleavages (23). To determine the gap size required for5�-end recognition, the RNase H cleavage pattern was compared usingsubstrates in which the same RNA was placed at different distancesdownstream fromPPT20, an RNaseH-resistant RNA containing nucle-otides �20 to �1 of the M-MuLV PPT sequence (Fig. 1 (37)). Thedistances between PPT20 and the downstream RNAs were either a nickor a gap of 1–5 bases. For M-MuLV reverse transcriptase, 5�-end-la-beled Md1 was used as the downstream RNA. In the No Upstreamsubstrate, cleavages occurred predominantly at sites D, E, and F inMd1(Fig. 8, lanes 1–5 and 36–40). Addition of upstream PPT20 to create anick reduced 5�-end-directed cleavages at these sites and promotedinternal cleavages at sites closer to the nick, such as sites A and B (lanes6–10). This cleavage pattern was identical to that observed using acontinuous RNA containing the same sequence but lacking the nick(23); together these data first suggested that a nick was insufficient todirect 5�-end-directed cleavages. Introduction of a 1- or 2-base gapbetween the upstream and downstreamRNAs decreased cleavage at the

A and B sites and increased cleavage at the E and F sites (Fig. 8, comparelanes 11–20 with lanes 6–10). The cleavage pattern with a 3-base gapmost closelymatched theNoUpstream substrate (compare lanes 21–25with 1–5 and 36–40). Interestingly, the highest extent of 5�-end-di-rected cleavageswas observed in substrateswith a gap size of 4 or 5 bases(lanes 26–35). Experiments performed using 3�-end-labeled Md1 con-firmed these results (data not shown).To test how a gap influences 5�-end-directed cleavages by HIV-1

reverse transcriptase, 5�-end-labeled Md10 was used as a downstreamRNA (Fig. 9). In the No Upstream substrate, the initial cleavages inMd10 occurred at sites H and I (lanes 1–5 and 36–40). Addition ofupstreamPPT20 decreased cleavage at sitesH and I and increased inter-nal cleavages at site E among others (lanes 6–10). For substrate with a1-base gap, cleavages closer to the RNA 5�-end such as site E decreasedwhile cleavages at sites H and I slightly increased (lanes 11–15). In the2-base and 3-base gap substrates, RNase H cleavages were most similarto those in substrate lacking upstreamRNA (compare lanes 16–25withlanes 1–5 and 36–40). Experiments performed with 3�-end-labeledMd10 and HIV-1 reverse transcriptase generated comparable results(data not shown).RNaseH assayswere also carried outwithM-MuLVorHIV-1 reverse

transcriptase and substrates containing downstream Md10 or Md1,respectively (date not shown). In both cases and similar to the datashown in Figs. 8 and 9, the pattern of RNase H cleavages in substrateswith a 2- or 3-base gap most clearly matched the No Upstreamsubstrate.

DISCUSSION

The ability to cleave the RNA strand of hybrids formed during reversetranscription is essential for retroviral replication. RNase H cleavagespositioned by an RNA 5�-end represent a very robust form of RNase Hactivity that likely provides an important role in general degradation ofthe viral genome. The data in this study demonstrate that multipledeterminants affect the specificity of RNA 5�-end-directed cleavage byretroviral RNases H.Previous work has shown that reverse transcriptase does not effi-

ciently utilize the RNA 5�-end at a nick to position 5�-end-directedcleavages (23). In this study, we addressed howmuch space between the3�- and 5�-ends of RNA is required to render the RNA 5�-end accessiblefor directing cleavage in the downstream RNA. By increasing the dis-tance between an upstreamRNA3�-end and a downstreamRNA5�-endin single nucleotide increments, we found that gaps of 2 or 3 basespermit 5�-end-directed cleavages at a level comparable to that observedfor a recessed, free RNA 5�-end. It seems likely that the binding ofreverse transcriptase to a recessed RNA 5�-end in a hybrid involvesrecognition of the discontinuity in the substrate structure at the junc-tion between the single strand and the duplex. A similar discontinuity isoffered by a typical primer-template, where the recessed end is aDNA3�terminus rather than an RNA 5�-end. Thus, perhaps the primer-tem-plate binding cleft and in particular the primer grip region (34) of thepolymerase domain positions the recessed RNA 5�-end of a hybrid for5�-end-directed cleavage by recognizing some of the same structuralfeatures presented by a primer terminus. In substrates containing a nickor a 1-base gap, the RNA 5�-end is obscured and RNase H cleavages arelimited to the internal model of cleavage. A substrate with a gap of 2 ormore bases offers a sufficient distance between the upstream and down-stream RNAs to allow the access and recognition required for the5�-end-directed mode of cleavage and, additionally, may offer contactsites in the upstream RNA that facilitate enzyme binding. During gen-




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FIGURE 5. Alignment of sequences flanking RNA5�-end-directed cleavage sites recognized byHIV-1 RNase H. The left column lists the names ofthe RNAs used in this analysis, and asterisks denotethose presented in prior studies. Cleavage sitesmapped using RNAs in this study are described inthe “Experimental Procedures.” The 28-nucleotideRNA is from Ref. 40. The 40-mer RNA from Ref. 25was used as a blunt-end hybrid, but a later studyhas shown that while the extent of cleavage isdiminished, the locations of 5�-end-directed cleav-age sites are not affected by a blunt end (40). RNAD is from previous studies (29, 32, 42). The 20-merRNA is from Ref. 45. In the center column, thesequence surrounding each cleavage site is given,with the location of the cleavage site representedas a gap. The right column gives the position ofeach cleavage site counting from the 5�-end of theRNA.




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eral degradation of the genome, it seems likely that gaps are importantto promote 5�-end-directed cleavages.To initially test how sequence might influence RNA 5�-end-directed

cleavages, RNase H assays were carried out with hybrid substrates thatcontained portions of the same sequence located at different positionsrelative to an RNA 5�-end. A comparison of the cleavages in these sub-strates (Fig. 10) reveals several interesting features for the 5�-end-di-rectedmode of cleavage that are shared between the reverse transcripta-ses of HIV-1 (arrows above sequences) and M-MuLV (arrows belowsequences). First, cleavages are not restricted to a discrete distance fromthe RNA 5�-end. Counting from the RNA 5�-end for HIV-1, strong ormedium cleavage sites were as close as between positions �13 and �14(Md1, Md4, Md7, and Md10) and as far as between positions �20 and

�21 (Md9). For M-MuLV, strong and medium cleavage sites rangedfrom between positions �13 and �14 (Md1, Md4, Md7, and Md10) tobetween positions �19 and �20 (Md9 and Md10). Second, the samesubstrate can be cleaved at multiple sites, and independent cleavagescan be distributed over a span of up to 8 nucleotides (for example, Md9and Md10). Third, cleavages occur at the same sites in the sequenceeven though these sites are located at different distances from the RNA5�-ends. As an example forHIV-1 RNaseH, siteG is cleaved in substrateMd1 between the 19th and 20th nucleotides, but this site is also cleavedwhen located 6 nucleotides closer to the RNA 5�-end in substrate Md7(indicated in Fig. 10). Fourth, some positions are consistently resistantto cleavage irrespective of distance from the RNA 5�-end, even whenlocated between other strong 5�-end-directed cleavage sites. Finally, the

FIGURE 6. Alignment of sequences flanking RNA5�-end-directed cleavage sites recognized byM-MuLV RNase H. The sequences are presentedas described in the legend to Fig. 5. Cleavage sitesmapped using RNAs in this study are describedunder “Experimental Procedures.”




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strong andmedium 5�-end-directed cleavage sites fall between the 13thand 20th nucleotides from the RNA 5�-end (Fig. 10, indicated with agray box). These data indicate that 5�-end-directed cleavage sites are notrandomly chosen and that both sequence and distance determine thecleavage positions of retroviral RNases H.Because some early studies suggested that sequence might influence

RNA 5�-end-directed cleavages (24, 29, 30), it is somewhat surprisingthat this possibility has not been investigated more thoroughly untilnow. Most likely this is because prior studies utilized a limited numberof RNAs and thus had very little variation in sequence (21, 29, 32,38–43). Also for several studies, the precise mapping of RNA 5�-end-directed cleavage sites was often difficult or not required by the exper-imental design (21, 29, 32, 46). Most recently, RNA 5�-end-directedcleavages have been examined with regard to the order of cleavage thatoccurs on an RNA/DNA hybrid with a recessed RNA 5�-end (38–40).From these studies, it was proposed that 5�-end-directed cleavages con-stitute an initial or primary cleavage �18 nucleotides from the RNA5�-end and are followed by independent, secondary cleavages that are 8to 9 nucleotides from the RNA 5�-end and occur at a slower rate.

Although our findings demonstrate that sequence is an importantdeterminant of 5�-end-directed cleavages, it remains to be investigatedhow sequence influences secondary cleavages.By mapping multiple 5�-end-directed cleavage sites in hybrids con-

taining RNAs with different sequences, we observed a statistically sig-nificant bias for specific nucleotides at positions flanking these sites. ForbothHIV-1 andM-MuLVRNasesH, A orC, but notG, was preferred atposition �1, and C or G, but not U, was preferred at position �2. Inaddition, HIV-1 RNaseHdisfavoredA at position�4. Precisely how thepreferred nucleotides facilitate RNase H cleavage is unknown, but it hasbeen proposed that the structures associated with some hybridsequences might affect cleavage specificity (35). Several features influ-enced by sequence are the base composition, the trajectory of the helicalaxis, and the width of the major and minor grooves (35, 47, 48).Although it is possible that the preferred nucleotides flanking a cleavagesite reflect interactions in the DNA strand instead of or in addition tothe RNA strand, the co-crystal structure of HIV-1 reverse transcriptaseand a PPT-containing RNA/DNA hybrid reveals potential hydrogen-bonding contacts between the�1 RNA base and Arg-448, and betweenthe �2 RNA base and Gln-475 (35) that might contribute to theobserved sequence preferences. Our observation that an A is disfavoredat the �4 position does not correspond to any base contacts in theco-crystal structure, but there are phosphate contacts from�4 to�9 inthe DNA strand and the RNase H primer grip region of the HIV-1enzyme (35). Notably, the �4 position in the co-crystal structure fallswithin an unusual structural deformation consisting of a region ofunpaired bases that may be PPT-specific and consequently may notreflect the binding situation for hybrids containing other sequences.Importantly, our data suggest that hybrid substrates containing the pre-ferred nucleotides at the most critical positions of �2 and �1 mayfacilitate the generation of co-crystals of reverse transcriptase withbound substrate in future crystallographic analyses.Very recently, the first co-crystal structure of a prokaryotic RNase H

with the hybrid substrate positioned in the enzyme active site has beenreported (49). The authors note that four residues (Asn-77, Asn-106,Gln-134, and Asn-105) donate hydrogen bonds to bases close to thescissile phosphate, and one residue (Arg-195) has a sequence-specificcontact with the DNA strand at the �5 G residue. This latter residue ishomologous to Arg-557 in HIV-1 reverse transcriptase, but thus far oursequence analyses of internal and 5�-end-directed cleavage sites havenot suggested a preference for C in the RNA strand at the �5 position(Ref. 23 and data not shown).Both the 5�-end-directed and internal modes of RNase H cleavage

prefer similar nucleotides at positions �1 and �2 for M-MuLV andpositions �1, �2, and �4 for HIV-1 (this work and Ref. 23). Internalcleavages also exhibit nucleotide preferences further upstream from thecleavage site (at positions �6 and �11 for M-MuLV and positions �7,�12, and �14 for HIV-1 (23), but no equivalent positions were identi-fied from the statistical analyses of 5�-end-directed cleavage sites. Theabsence of obvious nucleotide preferences further upstream of 5�-end-directed cleavage sites may derive from the nature of the upstreamsequences found in many of our hybrid substrates, where the first 8–10nucleotides of RNA sequence are dictated by the in vitro transcriptionvectors. Although these sequences may not offer the optimal nucleo-tides that would otherwise be preferred for 5�-end-directed cleavage,the specificity determinant contributed by binding the RNA5�-endmaysubstitute or compensate for additional upstream preferences. Thisconsideration suggests that preferred nucleotide positions locatedmostproximal to the cleavage site are the most important in determining thepositioning for both modes of RNase H cleavage.

FIGURE 7. Base preferences at nucleotide positions flanking strong and mediumRNA 5�-end-directed cleavages sites. The chi-square values of the nucleotide distribu-tion from positions �10 to �4 were calculated by comparison to overall base frequen-cies and plotted as a function of nucleotide position, with the cleavage site locatedbetween positions �1 and �1. A, analysis of the 46 sites for HIV-1 shown in Fig. 5. B,analysis of the 35 sites for M-MuLV shown in Fig. 6. The dashed line indicates the chi-square value of 11.34, which has a p value of 0.01. Nucleotide positions with scoresexceeding 11.34 are considered significant. The preferred nucleotides for each of thesignificant positions are indicated above the corresponding bar in uppercase (stronglypreferred) or lowercase (acceptable) letters.




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Aswould be predicted from the overlap between the preferred nucle-otide specificity of 5�-end-directed and internal cleavage sites, it appearsthat the 5�-end-directed sites are a subset of the possible internal sites

that are only cleaved when they meet certain distance constraints. Insupport of this prediction, all of the 5�-end-directed cleavage sites wehave observed thus far are also observed as internal cleavage sites on

FIGURE 8. Influence of gap size on 5�-end-di-rected cleavage of Md1 by M-MuLV reversetranscriptase. 5�-End-labeled Md1 was used as asubstrate without upstream RNA (No Upstream), asa substrate with PPT20 annealed immediatelyupstream (Nick), or as substrates with gaps of 1–5bases between the 3� terminus of upstream PPT20and the 5�-end of Md1 (1- to 5-base gap). Sub-strates were incubated with M-MuLV reverse tran-scriptase, and analyzed as described in Fig. 2. InMd1, cleavage site A is �2/�3, site B is �5/�6,and the other sites are as described in Fig. 3.

FIGURE 9. Influence of gap size on 5�-end-di-rected cleavage of Md10 by HIV-1 reverse tran-scriptase. 5�-End-labeled Md10 was used as a sub-strate without upstream RNA (No Upstream), insubstrate with PPT20 annealed immediatelyupstream (Nick), or in substrates with gaps of 1–5bases between the 3� terminus of upstream PPT20and the 5�-end of Md10 (1- to 5-base gap). Sub-strates were incubated with HIV-1 reverse tran-scriptase, and analyzed as described in Fig. 2. Prod-ucts resulting from cleavage at specific positionsin Md10 are indicated at the left, as described inFigs. 2 and 3. The smallest cleavage product (mostdistinct in lanes 7–10 below site E) corresponds tocleavage between the first and second nucleo-tides of Md10.




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longer RNA/DNA hybrids that do not require a nucleic acid end forpositioning (Ref. 23 and data not shown). For example, the predominant5�-end-directed sites in substrates Md1 through Md10 are also recog-nized as internal cleavage sites in longer RNAs containing the sequence�1 through �29 beyond the M-MuLV PPT origin (Figs. 1 and 2) (23).As indicated in Fig. 11A, only some of the possible internal cleavage sitesin the �1 to �29 sequence (indicated by arrows above the uppersequence) are recognized as 5�-end-directed sites in substratesMd1 andMd10 (indicated by vertical dashed lines). Importantly, the internal siteslocated close to the 5�- or 3�-ends of substrates Md1 or Md10 are notcleaved efficiently by the RNA 5�-end-directed mechanism because ofthe restrictions imposed by the distance from the RNA 5�-end.The internal mode of RNase H cleavage primarily recognizes sites

according to sequence (23). Our findings that a combination of anaccessible RNA 5�-end, proper sequence context, and an appropriatedistance from an RNA 5�-end are all determinants of RNA 5�-end-directed cleavages suggest a revision in the model for how retroviralRNases H carry out 5�-end-directed cleavages. In this model, 5�-end-directed cleavage sites are not chosen randomly at a measured distancefrom the RNA 5�-end. Instead, to be eligible for cleavage, a site mustconform to the preferred nucleotide sequence and the site must fallbetween a minimum and maximum range from an accessible RNA5�-end. We term this acceptable range the “cleavage window.” A bargraph depicting how frequently each position relative to the RNA5�-end is utilized in the strong and medium cleavage sites in Figs. 5 and6 reveals the breadth of the cleavage window (Fig. 11B). This graph

indicates that 5�-end-directed sites can occur at all positions within acleavage window between the 13th and 20th nucleotides from the RNA5�-end. As illustrated in Fig. 11A, the observed pattern of cleavages insubstratesMd1 andMd10 result from recognition of cleavage sites onlypositioned within this window.Our quantitative data with substrates containing 5�-end-labeled RNAs

(Fig. 4) also support the view that recognition of 5�-end-directed cleavagesites is optimal within a defined cleavage window. By defining that theextent of cleavage was significant if the cleavage product represented �5%of all bands, cleavages were optimal when the sites were located betweenthe 13th and 18th nucleotides for HIV-1, and between the 13th and 17thnucleotides for M-MuLV in these substrates. Because these experimentsused substrates with labeled RNA 5�-ends, these data were biased some-what against the more distal cleavages in the cleavage window.This cleavage window model accounts for the range of distances

observed in RNA 5�-end-directed cleavages assays. Although cleavagesites are often 15–19 nucleotides from the RNA5�-end (for example, seeRefs. 25, 27, 29, and 50–53), cleavages from 10 to 21 nucleotides fromthe RNA 5�-end have been reported in this study and others (29–32).These cleavage distances are broader than the 17 or 18 nucleotides thatseparate the polymerase and RNase H active sites in HIV-1 reversetranscriptase, based upon co-crystal structures of the enzyme withduplex DNA or RNA/DNA hybrids in which a DNA 3�-end fits in the

FIGURE 10. Comparison of HIV-1 and M-MuLV RNase H 5�-end-directed cleavages inthe sequences of RNAs Md1-Md10. The sequences of the 29-mer RNAs Md1 throughMd10 are aligned by the RNA 5�-ends to compare the positions of 5�-end-directed cleav-age sites. In each sequence, the extent of cleavage at a site is indicated as strong (largearrows) or medium (small arrows) for HIV-1 reverse transcriptase (above) or M-MuLVreverse transcriptase (below). As described under “Discussion,” the range of the closestand furthest independent 5�-end-directed cleavage sites is indicated by the positions ofthe bordering nucleotides from the RNA 5�-end, the position of site G in substrates Md1and Md7 is indicated, and the gray box highlights nucleotide positions �13 and �20 thatinclude the range of distances where the 5�-end-directed cleavages occur.

FIGURE 11. “Cleavage window” model for sequence and distance as determinantsof 5�-end-directed cleavage. A, the sequence of the M-MuLV region surrounding the�1/�1 site is shown, beginning at �20 and ending at �40, with the PPT underlined. Inthis sequence, the locations of the most prominent internal cleavage sites recognized byM-MuLV or HIV-1 reverse transcriptase (23) are indicated by arrows. Above each arrow isthe 5�- and 3�-nucleotide positions bordering the cleavage site, and the letter designa-tion given to that site. Site C is an internal cleavage site recognized by HIV-1 RNase H thatis not observed in 5�-end-directed cleavages, possibly because it is too close to the5�-end. The sequences of Md1 and Md10 are aligned below with vertical dashed linesindicating the positions of 5�-end-directed cleavage sites. The cleavage window thatdefines eligible sites for 5�-end-directed cleavage is indicated by the shaded box. B, afrequency distribution plot relative to specific positions that are used for 5�-end-directedcleavages for HIV-1 (black bars) or M-MuLV (gray bars), based upon the 46 sequencesshown for HIV-1 in Fig. 5 and the 35 sequences shown for M-MuLV shown in Fig. 6.




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polymerase active site (33–35). It is possible that contacts between thepolymerase domain and aDNA3�-primer terminusmore tightly anchorthe RNase H domain on the substrate, whereas an RNA 5�-end allowsmore flexibility in positioning. This would predict that the cleavagewindow for DNA 3�-end-directed cleavages is smaller than that forRNA 5�-end-directed cleavages, and experiments are underway to testthis possibility. It is also possible that the co-crystal structures reflect thedistance between the active sites when the enzyme initially bindsthe substrate, and that, in the case of RNA 5�-end-directed cleavages,the enzyme can slide on the substrate after the initial contacts with theRNA 5�-end are released or adjusted.Secondary RNase H cleavages have been proposed to occur inde-

pendently of primary, 5�-end-directed cleavages and result from an ini-tial binding and more extensive sliding of reverse transcriptase on itssubstrate (38–40). For M-MuLV, independent cleavages in this reportwere observed as close as between the 8th and 9th nucleotides from theRNA 5�-end (for example, site D in Md1). At this time, we cannotconclude whether these cleavages result from a secondary cleavage or a5�-end-directed mechanism. It may be that the definition of 5�-end-directed versus secondary cleavages is difficult to distinguish at sitescloser to the RNA 5�-end, and more experiments are required to deter-mine the nature of these cleavages.Afterminus-strand synthesis, extensivedegradationof theRNAgenome

is required to facilitateplus-strandsynthesis andstrand transfers, andmuchof this general degradation appears to proceed by the polymerization-inde-pendent mode of RNase H activity (20, 22, 25, 28). The remaining RNAtemplate that requires further degradation will likely contain nicks gener-ated during polymerization of the minus-strand. Because the RNA 5�-endat a nick does not efficiently promote 5�-end-directed RNase H cleavages,this formof RNaseH activity would be restricted to initially act at gaps, butwhether gaps of sufficient size are generatedduringminus-strand synthesisremains to be determined. Internal cleavages do not require positioning byan RNA or DNA end, so at least initially the polymerization-independentactivity of RNaseH can carry out internal cleavages. If recognition sites arelocated closely together and cleavedby the internalmode, thiswould creategaps that are sufficient for 5�-end-directed cleavages. Because 5�-end-di-rected cleavages are kinetically favored over internal cleavages (54), themultitude of possible internal cleavage sites combined with robust 5�-end-directed cleavages would together facilitate rapid and thorough degrada-tion of the retroviral genome.

Acknowledgments—We thank all members of the Champoux laboratory forhelpful discussions during the course of this work and especiallyHeidrun Inter-thal for critical review of the manuscript.

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Sharon J. Schultz, Miaohua Zhang and James J. ChampouxCleavages by Retroviral RNases H

-End-directed′Sequence, Distance, and Accessibility Are Determinants of 5

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