Discontinuous orsemi-discontinuous DNAreplication in Escherichia coli?Tzu-Chien V. Wang

SummaryThe postulate that a stalled/collapsed replication fork willbe generated when the replication complex encounters aUV-induced lesion in the template for leading-strandDNAsynthesis is based on the model of semi-discontinuousDNA replication. A review of existing data indicates thatthe semi-discontinuous DNA replication model is sup-ported by data from in vitro studies, while the discontin-uous DNA replication model is supported by in vivostudies in Escherichia coli. Until the question of whetherDNA replicates discontinuously in one or both strands isclearly resolved, any model building based on either oneof the two DNA replication models should be treated withcaution. BioEssays 27:633–636, 2005.� 2005 Wiley Periodicals, Inc.

Introduction

Faithful duplication of DNA is fundamental to all living

organisms. In prokaryotes, DNA replication is initiated at a

specific site on the DNA molecule, termed the origin of

replication, and then proceeds either in one or both directions

sequentially to the terminus. Replication of DNA is a semi-

conservative process, and is catalyzed by DNA polymerase in

the 50 to 30 direction. Because of the anti-parallel nature of a

DNA double helix, replication of DNA at a replication fork

cannot be continuous on both strands. A semi-discontinuous

model of DNA replication suggests that DNA synthesis is

continuous on the leading strand but is discontinuous on the

lagging strand. On the other hand, a discontinuous model of

DNA replication suggests that DNA synthesis is discontinuous

on both the leading and lagging strands. The question of

whether discontinuous DNA replication operates only for the

lagging strand or for both strands in Escherichia coli remains

unresolved.(1–3)

Depending on which DNA replication model is being used

by E. coli cells, the biological consequence of replicating

lesion-containing DNA can be very different (Fig. 1). If DNA

replicates semi-discontinuously, as popularly perceived, the

progress of a replication fork may be stalled by a UV-induced

DNA lesion in the template for leading-strand synthesis, since

normal DNA replication in E. coli is known to initiate at oriC,

and a primer may not be readily available to reinitiate DNA

synthesis downstream of a lesion in the leading-strand

template (Fig. 1A). On the other hand, if DNA replicates dis-

continuously in both strands, the progress of a replication fork

is unlikely to be stalled by a DNA lesion on either strand,

although the replicated DNA may contain daughter-strand

gaps (DSG) (Fig. 1B).

In thepast fewyears, there is a growing interest in the role of

recombination genes in the repair of stalled/collapsed replica-

tion forks.(4–8) In fact, it has been suggested that the major

function of recombination genes in rescuing E. coli cells from

UV damage is to repair stalled/collapsed replication forks,

rather than performing post-replication repair.(6,7) This new

idea challenges the paradigm that recombination genes play

an important role in the post-replication repair of UV-damaged

DNA. However, the importance of post-replication repair has

been defended in a recent review by Smith.(9) A careful

examination of the debate over whether recombination

functions primarily for the repair of stalled/collapsed replica-

tion forks or to perform post-replication repair in UV-irradiated

cells revealed a fundamentally unresolved question, i.e. does

replication of UV-induced DNA lesions in the leading-strand

template generate stalled/collapsed replication fork(5) or

DSG(10) (Fig. 1). Clearly, an answer to this issue depends on

our understanding of the replication mechanism in E. coli. In

this article, I will reexamine this question and update our

current understanding.

Discontinuous versus semi-discontinuous

DNA replication models

Theoretical consideration of the sizes of newly synthesized

DNA at a replication fork predicts that the nascent DNAs

synthesized by a discontinuous DNA replication model should

contain only low relative molecular mass (Mr) DNA fragments

when the two strands of DNA are separated, e.g. in an alkaline

sucrose gradient. In contrast, the nascent DNAs synthesized

by the semi-discontinuous DNA replication model are antici-

pated to contain two distinct sizes of DNA, the low Mr. DNA

fragments synthesized in the lagging strand and the high Mr.

DNA synthesized in the leading strand.

Department of Molecular and Cellular Biology, Chang Gung University,

Kwei-San, Tao-Yuan 333, Taiwan. E-mail: tcvwg@mail.cgu.edu.tw

DOI 10.1002/bies.20233

Published online in Wiley InterScience (www.interscience.wiley.com).

BioEssays 27:633–636, � 2005 Wiley Periodicals, Inc. BioEssays 27.6 633

Review articles

The first experimental test of these two models by Okazaki

et al.(11) led to the finding that nascent DNAswere all of lowMr

fragments if the time used to pulse-label nascent DNA was

very short. This led to the original proposal that DNA replicates

discontinuously in E. coli. Although many subsequent in vivo

data were also supportive of the two-strand discontinuous

replication model in E. coli (see Ref. 1 for review), this model

was met with skepticism for several simple reasons. First,

since the 30-OH of a growing DNA chain in the leading strand

can be used as a primer to continueDNAsynthesis, there is no

need to use de novo synthesized RNA primer to replicate the

leading strand discontinuously. Second, it is more energy-

consuming to synthesizeDNAbya discontinuousmechanism,

and it is not obvious why cells would evolve with such a

replication mechanism for leading-strand synthesis. There-

fore, with more logic than proof, the semi-discontinuous

replication model has been widely accepted.

Experimental data in favor of the

discontinuous and semi-discontinuous

replication models in E. coliEvidence supporting a semi-discontinuous replication model

inE. coli camemainly from an in vitro system that utilizes a cell

lysate prepared on a cellophane disk.(12,13) The DNA

synthesized in such a system is partitioned into two distinct

size classes when DNA joining is inhibited; short DNA pieces

(�9S) and longerDNAmolecules (�38S). The amount of DNA

synthesized in each of these two size classes is about equal,

and the sequences of theDNAmolecules in each class are not

complementary, but the sequences in one class are comple-

mentary to the sequences in the other class. Only the short

DNA pieces seem to be synthesized discontinuously, since

their synthesis requires functional primase, DnaG.(14) Such an

asymmetric DNA synthesis in vitro is consistent with a semi-

discontinuous DNA replication model.

Most of the in vivo data, on the other hand, suggest that

DNA replication in E. coli is discontinuous on both strands.

Following the original observation for discontinuous DNA

replication in several wild-type strains of E. coli,(11) the ques-

tion of whether discontinuous DNA replication operates only

for the lagging strand or for both strands had been examined

by many investigators. Although there had been a few reports

that semi-discontinuousDNAsynthesiswasobserved in some

E. coli strains,(15,16) it was found later that the method used to

terminate the pulse by these authors allowed the joining of

nascent DNA fragments.(17) When care was taken to properly

terminate the pulse, it was found that all of the wild-type E. coli

strains examined synthesized DNA discontinuously on both

strands.(17) Additional in vivo evidence supporting a discontin-

uous DNA replication model came from studies with tempera-

ture-sensitive ligase (lig) mutants,(18–21) and with DNA

polymerase I (polA) mutants,(21–24) i.e. after pulse labeling

these mutants with [3H]thymidine, virtually all of the nascent

DNA was found in short DNA chains. However, since ligase

and DNA polymerase I are also required for the completion of

several DNA repair processes (such as mismatch repair and

excision repair), these results can also be interpreted to

suggest that DNA replication is really continuous in the leading

strand, but the observed strand interruptions resulted from

some ongoing repair process.

Among the DNA repair processes that can introduce

breaks in the DNAmolecule are: (1) nucleotide excision repair,

which employs the UvrABC excinuclease (encoded by the

uvrA, uvrB, uvrC genes) to excise a wide variety of DNA

damage,(25,26) (2) mismatch repair, which corrects mis-

matched base-pairs in the newly synthesized DNA strand,(27)

Figure 1. Diagram of hypothetical

structures following the replication of

lesion-containing DNA. A: A semi-dis-

continuous replication model predicts

that the progress of a replication fork

(RF) is stalled by a UV-induced DNA

lesion (shown as filled square in the

figure) in the template for leading-strand

synthesis, but may not be stalled by a

UV-induced DNA lesion in the template

for lagging-strand synthesis. A daugh-

ter-strand gap (DSG) is thought to be

produced in the replicated DNA. B: Adiscontinuous replication model pre-

dicts that the progress of a replication

forkwill not be stalledbyaDNA lesionon

either strand, and both strands of the

replicated DNA should contain DSG.

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634 BioEssays 27.6

and (3) base-excision repair, which employs a specific

glycosylase for the removal of an altered base by cleavage

at theglycosidic bond, creatinganapurinic or apyrimidinic (AP)

site.(28) These AP sites are subsequently removed and repair-

ed by the combined action ofAPendonuclease, deoxyribopho-

sphodiesterase,DNApolymeraseand ligase.(29) The inhibition

of DNA uracil–DNA glycosylase by an ung mutation did not

eliminate the accumulation of short DNA chains in a polA

strain.(30) When themutations inactivating nucleotide excision

repair (uvrB5), mismatch repair (mutL218 or mutS215), and

base-excision repair of DNA uracil (ung-1 or ung-152) were

introduced into a temperature-conditional lig-7 strain, the bulk

of nascent DNA synthesized at 428C were smaller than 2 kb

(i.e. about the size of Okazaki fragments), indicating that the

apparent discontinuous DNA replication in a lig-7 strain is not

the result of nucleotide excision repair, mismatch repair or the

base-excision repair ofDNAuracil.(31) Inactivation of twomajor

AP endonucleases, ExoIII and EndoIV, by introducing the

del(xth-pnc)90 and nfo-1::kan mutations into a uvrB5 lig-7

strain also did not eliminate the accumulation of short DNA

chains when the uvrB5 lig-7 del(xth-pnc)90 nfo-1::kan cells

were grown at 428C.(32) These results indicate that the

apparent discontinuous DNA replication in E. coli lig-7 strains

cannot be due to known DNA-repair processes.

Several possible explanations may be offered to account

for the apparent discontinuousDNA replication in a lig-7 strain:

(1) leading-strand DNA synthesis may be disrupted in a lig-7

strain at the non-permissive temperature, therefore, the

observed nascent DNA fragments may all be derived from

Okazaki fragments synthesized on the lagging strand, or

(2) there are yet unidentified DNA repair process, which

can generate breaks in the nascent leading strand. The

possibility that the accumulated Okazaki fragments in a lig-7

strain may be derived only from lagging-strand synthesis

has been tested using strand-specific DNA probes. It was

shown that the Okazaki DNA fragments obtained from lig-7

cells grown at 428C or from wild-type cells had equal

amounts of leading-strand and lagging-strand sequences.(32)

Therefore, unless there are as yet unidentified DNA repair

processes that can generate the apparent discontinuity in the

leading strand, existing in vivo data are in favor of the

discontinuous DNA replication model originally proposed by

Okazaki et al.(11)

Finally, for the discontinuous DNA replication model to

operate inE. coli cells, onewould predict that RNA primers are

synthesized on both leading and lagging strands. Therefore,

the DnaG primase-binding sites should be present on both

strands, and be spaced roughly at a distance equal to the size

of average Okazaki DNA fragments. The complete genome

sequence of E. coli K-12 is available.(33) Sequence analysis

indicates that DnaG primase-binding sites are abundant on

both strands, and their spacing is consistent with Okazaki

fragment sizes.(33) Although there is no direct proof implicating

these sequences in discontinuous DNA replication, the

sequence data provide additional support that discontinuous

DNA replication can occur in E. coli.

Conclusions

Thevalidity of aDNA replicationmodel should be judgednot by

its popularity and logic, but rather from the experimental facts

that support it. While most of the in vitro studies indicate a

semi-discontinuous DNA replication model, most of the in vivo

data favor a discontinuous replication model in E. coli. The

asymmetric DNA synthesis observed in vitro(12,13) may

be caused by the high concentration of NMN (nicotinamide

mononucleotide) and/or by lysis of cells that lead to semi-

discontinuous replication. On the other hand, the discontin-

uous DNA replication observed in vivo may be caused by an

unknown repair process,which produces the apparent discon-

tinuity for the nascent DNA synthesized on the leading-strand.

Clearly, there is a need to resolve the contradictory results

between the in vitro and in vivo studies before we can know for

sure whether E. coli replicates DNA discontinuously or semi-

discontinuously.

A very important question that needs to be addressed is

whether RNA primers are synthesized on one or both strands.

For the discontinuous DNA replication model to operate in

E. coli, onewould predict that RNA primers are synthesized on

both the leading and lagging strands. Although the DnaG

primase-binding sites are abundant on both strands, and their

spacing is consistent withOkazaki fragment sizes,(33) we don’t

know if the sites located on the leading-strand template are

actually used to synthesize RNA primers. If these sites are

used, does this primosome complex differ from the DnaB–

DnaG primosome complex that is thought to participate in the

synthesis of lagging-strandRNA?Future experiments addres-

sing these issues should help to resolve the question of

whether discontinuous DNA replication operates only for the

lagging strandor for both strands inE. coli. Until this question is

clearly resolved, anymodel building based on either one of the

two DNA replication models should be treated with caution.

The stalled/collapsed replication forkmodel recently proposed

for UV-irradiated cells(5–7) is based on a semi-discontinuous

DNA replication model, which is not supported by the existing

in vivo data in E. coli. Therefore, interpretations of results

based on such a model should be evaluated with the

understanding that the question of discontinuous or semi-

discontinuous DNA replication in E. coli remains an issue of

controversy.

Acknowledgments

Wewish to thank Drs. Kendric C. Smith and Neil, J. Sargentini

for their helpful suggestions. This work is supported by

National Science Council Research Grant NSC 92-2311-

B182-002 and Chang Gung Medical Research Grant BMRP

018 of Taiwan.

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BioEssays 27.6 635

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