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Chen Yonggang
Zhejiang University
Schools of Medicine
Biochemistry
DNA Replication
.
DNA Replication-Conservation of Information
• DNA replication must be carried out every time a cell divides
• Procaryotic growth involves cell division• Mitosis in eucaryotes involves cell division• DNA replication is template driven and
synthesizes DNA in a semi-conservative manner
dNTP + DNAn DNAn+1 +PPi
DNA is replicated in a semi-conservative manner
• Messelson & Stahl showed, using 15N-labelled DNA that the products of replication had intermediate density
McKee 18.2
15Nstrand A strand B
15N14N
15N 15N14N 14N14N 14N
15N 15N14N14N14N14N 14N14N
Each separated DNA strand is duplicated to give two
new double helices.
DNA semiconservative replication
• Each DNA strand serves as a template for the synthesis of a new strand, producing two new DNA molecules , each with one new strand and one old strand, this is semiconservative replication.
The process which appeared simple initially is complex
• Replication occurs just prior to cell division• The E coli chromosome is a single circular
DNA double helix associated with proteins in a nucleoid
• The E coli chromosome is negatively supercoiled and thus is quite compact and inaccessible
McKee 17.16
To allow the replication to occur supercoiling must be relaxed
• A type I (single strand breaking) topoisomerase cleaves and relaxes the negative supercoiling ahead of the replication complex
• The topoisomerase has a central hole through which the double helix passes. An intermediate is an enzyme-linked 3’OH
• dnaA is displaced providing access for the next component needed for replication
The first step in replication involves oriC
• OriC is a 245 bp region, the origin of E coli replication
• OriC contains 3 tandem repeats of a 13 bp sequence beginning in GATC and rich in AT bp
• These repeats are weakly H-bonded and serve to provide 4 binding sites for a protein, dnaA, a start signal for replication
• Replication proceeds in two directions - bidirectional
dnaA allows binding of two other important proteins
• dnaB is a DNA helicase which carries out the ATP- driven unwinding of the DNA double helix
• dnaC is an important accessory protein which binds and is soon released
• Together the three proteins utilize ATP to bend and separate the two strands of the bacterial chromosome
SSB, a single strand binding tetramer stabilizes the initiation complex
McKee 18.7
DNA replication involves many enzymes
• An RNA primase binds to the SSB stabilized melted helix
• Since DNA polymerases require a primer and only extend that primer, the RNA primase (dnaG) in association with other proteins (primasome) synthesizes a 5-7 nucleotide primer using information from the template strand
At the replication fork two strands are managed differently
• The 5’ end of the primer(leading strand) is extended continuously by DNA polymerase III in a 5’-3’ direction by dNTPs
• The primer on the lagging strand is also extended 5’-3’ by DNA polymerase III, in a discontinuous manner
• Thus primer is made once on the leading strand and every 1000 nucleotides on the lagging strand
The replication fork
McKee 18.6
DNA Polymerase III holoenzyme contains 10 distinct types of subunits
• DNA Polymerase III is the primary replicase in E. coli
• It has polymerase and 3’-5’ exonuclease activities• It functions as a dimer• It has great fidelity, only 1 error in 1010 bp• It is highly processive, sticking to the DNA for the
entire trip through the chromosome• It has a rapid biosynthetic rate, synthesizing 1000
nt/sec
The Pol III synthesizes DNA from dNTPs
McKee 18.3
The Dimer moves in one direction and synthesizes 5’-3’
• The leading strand is synthesized by addition of 5’dNTPs in response to the template
• The looped lagging strand is synthesized in Okazaki fragments using 5’dNTPs
• The lagging strand must be pieced together using DNA Polymerase I
DNA polymerase III forms phosphodiester bonds
• 2’-deoxynucleoside 5’ triphosphates are the activated intermediates needed for synthesis
• Information from the parental strand provides the information for 5’-3’ synthesis (parental strand is read 3’-5’)
• Thus each parental strand serves as the template for synthesis of a complementary strand
Synthesis of a phosphodiester bond
OBASE1OP
O
O
CHAIN
OH
P
O
O
O O OP
O
O
OBASE2OP
O
O
OH
..nucleophilic attack
OBASE1OP
O
O
CHAIN
O
OBASE2OP
O
OH
O
new phospho-diesterbond
DNA Polymerase III is at the center of Replication
McKee 18.8
Top-down view of replication
While DNA polymerase III does the replicating, DNAP I cleans up• Pol I(100kd) is a monomer of about 10%
the size of Pol III(900kd)
• It has three activities– It is a DNA polymerase– It is a 3’-5’ exonuclease– It is a 5’-3’ exonuclease
• It is a processing and proofreading enzyme
The three activities are on one polypeptide
• The larger fragment(Klenow fragment) of 67kd contains the polymerase and the 3’-5’ exonuclease
• The smaller, 36kd contains the 5’-3’ exonuclease activity
As Pol III finishes, Pol I goes to work
• The 5’-3’ exonuclease removes the RNA primer
• The polymerase synthesizes DNA to fill the gap
• Errors in Pol III synthesis are removed by 3’-5’ exonuclease
Supercoiling was taken out by dnaB, DNA gyrase replaces it
• Following synthesis of the strands, excision of RNA, replacement by DNA using Pol I, the supercoiling can be reinstated
• DNA gyrase, an ATP- linked, energy requiring enzyme introduces negative supercoils to restore the original twist in the leading strand
DNA ligase seals the Okazaki fragments and the completed
double helical DNA• This energy requiring enzyme uses NAD as the
activated AMP donor in this reaction• Pyrophosphate cleavage drives the reaction to
completion• Termination occurs at a ter region and is mediated
by a binding protein TBP• A type II(double stranded) topoisomerase is
probably involved in helix dissociation
Eucaryotic Replication is similar to that of procaryotes
• Both have initiation, elongation and termination phases and are bidirectional
• Both involve multiple DNA polymerases• Both involve multiple copies of the primary
replicase which replicates strands differently• Replication rate is slower, but replication is rapid
due to multiple replicons• Both require topoisomerases to unwind and
rewind the DNA• Both require ligases
Eucaryotic replication is distinct from that of procaryotes
• There are 5 polymerases• The chromosomes are linear• There are multiple ori, and replication units• Replication only occurs during the S phase
of the cell cycle• Telomeres restrict the number of times a
replicon can be expressed
Initiation of replication occurs at multiple ori
• A large complex of proteins assembles at an ori (Origin Recognition Complex-ORC)
• Details not for testing– A complex with helicase activity must bind and be
activated– Replication Protein A (RPA ) binds and separates the
strands(like SSB in E.coli)– RFC(replication factor C– a clamp loading factor) and
PCNA(proliferating cell nuclear antigen) allows binding of Pol to both the leading and lagging strand
Binding of initiation factors to the lagging strand differs
• Pol is the main eucaryotic replication polymerase (Details not for testing)– Replication protein A(RPA) binds to the single strands
– Pol a and a primase complex binds to the lagging strand
– An RNA primer and 15-30 dNTPs are synthesized
• Pol binds and replicates one nucleosomes worth of Okazaki fragment
Finishing and sealing of the lagging strand is different
• Specific protein factors are important in finishing up replication (details not for testing)– DNA polymerase remove the primers and DNA
polymerase excise errors
– Topoisomerases induce supercoiling
– DNA ligase seals the breaks
– Chromosomes segregate
• Replication bubbles merge• Telomeres determine the end of replication
Telomeres are GC rich self-complementary sequences at
chromosome ends
• Telomerase maintains the telomeres
• Telomeres are repeat structures with a terminal loop
• At each replication the telomeres are modified using an integral RNA template
• Loss of telomeres limits replication
• Cancer cells lose control of their telomeres