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Semi consevative SSB (single-Stranded binding protein) Bidirectional Sliding ringsOrigin of Replication (Ori C) Clamp, clamp loaderGrowth fork Dna A, Dna B (Hexamer)Dna CLeading StrandLagging StrandOkazaki FragmentsDNA Poly I, II, III5’----3’ Polymrase3’----5’ Exonuclease5’----3’ ExonucleaseKlenow Fragment Proof readingRNA PrimerPrimasePrimosome (Primase+ Helicase)DNA Ligase
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HistoneDNA Polymerase α,β,γ, δ, ε)ORC (Origin recognition complex)ARS (Autonomously Replicating Sequence)Core element (11 bp)PCNA (clamp)RFC ( Clamp loader)RNase H, Fen 1
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Model of initiation of replication at E. coli oriC. The 9-mers and 13-mers are the repetitive sequences. Multiple copies of DnaA protein bind to the 9-mers at the origin and then “melt” (separate the strands of) the 13-mer segments. The sole function of DnaC is to deliver DnaB, which is composed of six identical subunits, to the template. One DnaB hexamer clamps around each single strand of DNA at oriC, forming the prepriming complex. DnaB is a helicase, and the two molecules then proceed to unwind the DNA in opposite directions away from the origin
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OriC has a length of 245 bp. It contains a tandem array of three nearly identical 13-nucleotide sequences (green) and four binding sites (yellow) for the dnaA protein. The relative orientations of the four dnaA sites are denoted by arrows.
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Consensus sequence of the minimal bacterial replication origin based on analyses of genomes from six bacterial species. Similar sequences constitute each origin; the 13-bp repetitive sequences (orange) are rich in adenine and thymine residues. The 9-bp repetitive sequences (brown) exist in both orientations; that is, the lower-right sequence, read right to left, is the same as that of the upper-left sequence, read left to right. These sequences are referred to as 13-mers and 9-mers, respectively. Indicated nucleotide position numbers are arbitrary
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DNA is not synthesised in the 3′ to 5′ direction, as erroneously shown on the left-hand strand
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Helicase Structure. The bacterial helicase PcrA comprises four domains: A1, A2, B1, and B2. The A1 domain includes a P-loop NTPase fold, whereas the B1 domain has a similar overall structure but lacks a P-loop and does not bind nucleotides. Single-stranded DNA binds to the A1 and B1 domains near the interfaces with domains A2 and B2.
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Enzyme Subunits 3′→5′Exo
5′→3Exo′
Function
Bacterial DNA polymerases
DNA polymerase I 1 Yes Yes DNA repair, replication
DNA polymerase III At least 10 Yes No Main replicating enzyme
Eukaryotic DNA polymerases
DNA polymerase α 4 No No Priming during replication
DNA polymerase γ 2 Yes No Mitochondrial DNA replication
DNA polymerase δ 2 or 3 Yes No Main replicative enzyme
DNA polymerase ε At least 1 Yes No Required for detection of DNA damage during genome replication
DNA polymerase κ 1 or 2? ? ? Required for attachment of cohesin proteins which hold sister chromatids together until the anaphase stage of nuclear division
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Two metal ions (typically, Mg2+) participate in the DNA polymerase reaction. One metal ion coordinates the 3′-hydroxyl group of the primer, whereas the phosphate group of the nucleoside triphosphate bridges between the two metal ions. The hydroxyl group of the primer attacks the phosphate group to form a new O-P bond
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DNA polymerases donate two hydrogen bonds to base pairs in the minor groove. Hydrogen-bond acceptors are present in these two positions for all Watson-Crick base pairs including the A-T base pair shown.
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Proofreading. The growing polynucleotide chain occasionally leaves the polymerase site of DNA polymerase I and migrates to the exonuclease site. There, the last nucleotide added is removed by hydrolysis. Because mismatched bases are more likely to leave the polymerase site, this process serves to proofread the sequence of the DNA being synthesized.
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Proposed Architecture of DNA Polymerase III Holoenzyme
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Core Enzyme: subunit: DNA polymeraseε subunit:3’---5’ exonucleaseθ subunit: Stimulate ε exonuclease
Holoenzyme:β subunit:sliding clampδ subunit : binds to β
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Shape Selectivity. The binding of a nucleoside triphosphate (NTP) to DNA polymerase induces a conformational change, generating a tight pocket for the base pair consisting of the NTP and its partner on the template strand. Such a conformational change is possible only when the NTP corresponds to the Watson-Crick partner of the template bas
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Structure of the Sliding Clamp. The dimeric β2 subunit of DNA polymerase III forms a ring that surrounds the DNA duplex. It allows the polymerase enzyme to move without falling off the DNA substrate
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DNA Polymerase Structure. The first DNA polymerase structure determined was that of a fragment of E. coli DNA polymerase I called the Klenow fragment. Like other DNA polymerases, the polymerase unit resembles a right hand with fingers (blue), palm (yellow), and thumb (red). The Klenow fragment also includes an exonuclease domain
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Helicase Structure. The bacterial helicase PcrA comprises four domains: A1, A2, B1, and B2. The A1 domain includes a P-loop NTPase fold, whereas the B1 domain has a similar overall structure but lacks a P-loop and does not bind nucleotides. Single-stranded DNA binds to the A1 and B1 domains near the interfaces with domains A2 and B2
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Helicase Mechanism. Initially, both domains A1 and B1 of PcrA bind single-stranded DNA. On binding of ATP, the cleft between these domains closes and domain A1 slides along the DNA. On ATP hydrolysis, the cleft opens up, pulling the DNA from domain B1 toward domain A1. As this process is repeated, double-stranded DNA is unwound.
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Conserved Residues among Helicases. A comparison of the amino acid sequences of hundreds of helicases revealed seven regions of strong sequence conservation (shown in color). When mapped onto the structure of PcrA, these conserved regions lie along the interface between the A1 and B1 domains and along the ATP binding surface
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The images show the crystal structure of Klenow fragment (top row) and Klenow fragment complexed with DNA (bottom row) solved to 2.8 and 3.2 Å,
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The DNA polymerase III holoenzyme consists of the core enzyme, the b sliding clamp and the clamp-loading complex.
the b sliding clamp The above images show different views of the beta subunit dimer. The central hole is evident in all three images. The top right-hand image shows a detailed model of the two polypeptides. The top left -hand image shows the polypeptide backbones of the two subunits. The bottom left hand image shows the secondary structure of the polypeptides. The interior of the dimer is lined with alpha-helices; the exterior is formed by beta sheets. The bottom right hand image shows how the beta polypeptide can form a sliding clamp on DNA.
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The clamp-loading complex consists of the delta (δ) (38.7 kD) and delta (δ') prime (36.9 kD), chi (χ) (16.6 kD) and psi (ψ) (15.2 kD) subunits and either or both of the gamma (γ) subunit (68.4 kD) and the tau (Τ)
subunit (71.1 kD). Both the gamma (γ) subunit and the tau(Τ) subunit are 'motor' ATPases. The δ subunit
binds to the β subunit and, in concert with the δ' and γ subunits and with ATP, it catalyses the opening of
the β dimer to permit passage of DNA.
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DNA polymerase
I II III
Gene for the polymerase subunit
polA polB polC
No. of subunits 1 > 4 > 10
Proofreading (3' -> 5') exonuclease activity?
yes yes yes
5' -> 3' exonuclease activity?
yes no no
polymerization rate (nucleotides added per second)
16-20 5 - 10 250-1000
processivity (nucleotdes added before dissociation)
low(3-200)
high(10,000)
very high(500,000)
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DNA Replication. The formation of a phosphodiester bridge is catalyzed by DNA polymerases.
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DNA Polymerase
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Table 2. Eukaryotic DNA polymerases
PolFamily Subunit composition (kDa)1 Function(s)2
Pol a B 166 (167) Replication, repair (DSBR), telomere maintenance,
66 (79) S phase checkpoint control
59 (62)
50 (48) Priming in replication
Pol d B 124 (125) Replication, repair (MMR, BER, NER, DSBR), recombination, telomere maintenance, S phase checkpoint control
51 (55)
51 (40)
12
Pol e B 262 (256) Replication, repair (BER, NER, DSBR), recombination, transcriptional silencing, S phase checkpoint control
60 (79)
17 (23)
12 (22)
Pol z B 353 (173) Translesion synthesis
24 (29)
Pol g A 140 (144) Mitochondrial DNA replication
55
Pol q A 198 Interstrand cross-link repair?
Pol b X 38 Repair (BER)
Pol l X 63 (68) Meiotic repair
Pol m X 55 Somatic hypermutation
Pol σ1 (Trf4) X 57 (66) Establishment of sister chromatid cohesion
Pol σ2 (Trf5) X 60 (73) Establishment of sister chromatid cohesion
Pol h Y 78 (72) Translesion synthesis
Pol i Y 80 Translesion synthesis, mutagenesis?
Pol k Y 99 Translesion synthesis
Rev1 Y 138 (112) Translesion synthesis
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Replication protein Eubacteria1 Eukaryotes
Origin recognition & unwinding4
DnaA* ORC*
Licensing/helicase loading DnaC* Cdc6/Cdc18
Licensing, helicase6 DnaB Mcm2-3-4-5-6-7*
Primase DnaG Pol α-primase
ssDNA binding & stabilization
SSB RPA
Processivity clamp8 β -subunit of Pol III
PCNA
Clamp loader9 γ -complex of Pol III*
RFC*
DNA polymerase PolC, PolA(Pol III, Pol I)
PolB(Pol α, Pol δ, Pol ε)
Maturation nuclease RnhA Fen1
Ligase Ligase Ligase I
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Proliferating cell nuclear antigen
Proliferating cell nuclear antigen
Proliferating cell nuclear antigen
commonly known as PCNA, is a protein that acts as a processivity factor for DNA polymerase delta in eukaryotic cells. It achieves this processivity by encircling the DNA, thus creating a topological link to the genome. It is an example of a DNA clamp.
Since DNA polymerase delta is involved in resynthesis of excised damaged DNA strands during DNA repair, PCNA is important for both DNA synthesis and DNA repair.[7] PCNA is also involved in the switch to DNA damage bypass translesion synthesis
Proliferating Cell Nuclear Antigen (Trimer)
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DNA Polymerase α
1-involved in initiation2-the DNA-replicating enzyme3-synthesizes an RNA primer then adds dNTPs4-a complex of four subunits6-50-kD and 60-kD are primase subunits;180-kD subunit DNA polymerase7-synthesizes 8-10 nt RNA primers, then adds DNA to the RNA primers8-low processivity of DNA synthesis (200 nt)9-has no 3’ -5’ exonuclease activity (proofreading), yet has high fidelity
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DNA Polymerase δ (Delta)
1-the principal DNA polymerase in eukaryotic DNA replication2-has 3’-5’ exonuclease activity3-consists of a 125 kdal and a 50 kdal subunit4-the 50 kd subunit interacts with PCNA (Proliferating Cell Nuclear Antigen)5-is highly processive when in association with PCNA
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DNA Polymerase ε
1-required for replication, but its role is unclear2-may substitute for DNA polymerase δ (Delta) in lagging strand synthesis
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Additional Proteins Involved in Eukaryotic DNA Synthesis
PCNA (Proliferating Cell Nuclear Antigen)-confers high processivity to DNA Polymeraseδ -the eukaryotic counterpart of the 2 Sliding Clamp of E. coli-PCNA also encircles the double helix, but is a homotrimer of 37 kD subunits
RPA (Replication Protein A)-ssDNA-binding protein that facilitates the unwinding of the helix to create two replication forks-the eukaryotic counterpart of the SSB protein of E. Coli
RFC (Replication Factor C)-the eukaryotic counterpart of the complex Clamp Loader of E. coli
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Leading strand synthesis
1) Starts with the primase activity of DNA Pol to lay down a primer2) lays down an RNA primer, then the DNA pol component of Pol adds a stretch of DNA3) RFC assembles PCNA at the end of the primer4) PCNA displaces DNA Pol .5) DNA polymerase δ (Delta) binds to PCNA at the 3’ ends of the growing to carry out.Highly processive DNA synthesis(Polymerase Switching)
RFC Mediates Polymerase Switching1) Assembly of PCNA2) Removes DNA Pol 3) Addition of DNA Pol δ
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Lagging strand synthesis
1) starts off the same way as leading strand synthesis
2) RNA primers synthesized by DNA polymerase á every 50 nt and consist of 10-nt RNA + 10-20-nt DNA
3) polymerase switching as before to extend the RNA-DNA primers to generate Okazaki fragments
4) when the DNA Pol ä approaches the RNA primer of the downstream Okazaki fragment, RNase H1 removes all but the last RNA nucleotide of the RNA primer
5) the FEN1/RTH1 exonuclease complex removes the last RNA nucleotide6) DNA Pol ä fills in the gap as the RNA primer is being removed
7) DNA ligase joins the Okazaki fragment to the growing strand
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