Structure and function of the bacteriophage T4 DNA polymerase holoenzyme

Young, Mark C.; Reddy, M. Jaipal; Hippel, Peter H. von

doi:10.1021/bi00152a001

Cited by 108 publications

(79 citation statements)

References 116 publications

(209 reference statements)

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“…10C). In contrast, gp32, which stimulates specifically DNA synthesis catalyzed by T4 pol (31,51,60), endows the polymerase with a strand displacement activity, in agreement with previous reports (41,53), probably by direct protein-protein interactions (23,51,52) cific protein-protein interactions, and differences in their processivity. A low processivity would favor heteroduplex formation by promoting the dissociation of the polymerase during replication of the first repeat, whereas high processivity would favor synthesis of parental molecules by preventing the dissociation of the polymerase.…”

Section: Figsupporting

confidence: 92%

“…21 and 22), whereas T4 pol (gp43) is associated with gp45 (the processivity factor) and gp44-gp62 (the clamp loader; Ref. 23). The two polymerase subunits are far less processive than their holoenzyme counterparts (22,23).…”

Section: Figmentioning

confidence: 99%

“…23). The two polymerase subunits are far less processive than their holoenzyme counterparts (22,23). E. coli SSB stimulated DNA synthesis catalyzed by pol II, as expected from previous reports (22,49,60,61), maybe by direct protein-protein interactions (62), but did not affect its capacity to slip or its strand displacement activity (Ref.…”

Section: Figmentioning

confidence: 99%

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Replication Slippage of Different DNA Polymerases Is Inversely Related to Their Strand Displacement Efficiency

Canceill¹,

Viguera²,

Ehrlich³

1999

Journal of Biological Chemistry

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Replication slippage is a particular type of error caused by DNA polymerases believed to occur both in bacterial and eukaryotic cells. Previous studies have shown that deletion events can occur in Escherichia coli by replication slippage between short duplications and that the main E. coli polymerase, DNA polymerase III holoenzyme is prone to such slippage. In this work, we present evidence that the two other DNA polymerases of E. coli, DNA polymerase I and DNA polymerase II, as well as polymerases of two phages, T4 (T4 pol) and T7 (T7 pol), undergo slippage in vitro, whereas DNA polymerase from another phage, ⌽29, does not. Furthermore, we have measured the strand displacement activity of the different polymerases tested for slippage in the absence and in the presence of the E. coli single-stranded DNA-binding protein (SSB), and we show that: (i) polymerases having a strong strand displacement activity cannot slip (DNA polymerase from ⌽29); (ii) polymerases devoid of any strand displacement activity slip very efficiently (DNA polymerase II and T4 pol); and (iii) stimulation of the strand displacement activity by E. coli SSB (DNA polymerase I and T7 pol), by phagic SSB (T4 pol), or by a mutation that affects the 3 3 5 exonuclease domain (DNA polymerase II exo ؊ and T7 pol exo ؊ ) is correlated with the inhibition of slippage. We propose that these observations can be interpreted in terms of a model, for which we have shown that high strand displacement activity of a polymerase diminishes its propensity to slip.Misalignment of two DNA strands during replication can lead to DNA rearrangements such as deletions or duplications of varying lengths ranging from several nucleotides to entire genes. This process, designated replication slippage (as well as copy-choice recombination), has been suspected for a long time to occur both in prokaryotes and eukaryotes between repeated DNA sequences. The process is thought to encompass the following steps: (i) copying of the first duplication by the replication machinery, (ii) replication pausing and dissociation of the polymerase from the newly synthesized end, (iii) unpairing of the newly synthesized strand and its pairing with the second duplication, and (iv) resumption of the DNA synthesis. A heteroduplex is thus formed, containing one parental and one recombinant strand, which are separated by a second round of replication.Replication slippage has been widely proposed as a probable mechanism of genome rearrangements, such as deletions between short duplications in bacteria (1-3), yeast (4), and mammalian mitochondria (5) or deletions between long tandem repeats in Escherichia coli (6 -8), as well as microsatellite instability (for reviews see Refs. 9 -12). Direct evidence for the slippage has been obtained in vivo, in E. coli (13), and in vitro (14). In the latter study, it was shown that E. coli DNA polymerase III holoenzyme (pol III HE), 1 the enzyme that replicates the cell chromosome (for review see Ref. 15), was able to slip, which is of particular significance in vie...

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Section: Figsupporting

confidence: 92%

Section: Figmentioning

confidence: 99%

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Replication Slippage of Different DNA Polymerases Is Inversely Related to Their Strand Displacement Efficiency

Canceill¹,

Viguera²,

Ehrlich³

1999

Journal of Biological Chemistry

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“…Factors responsible for both the initiation and elongation of DNA have been extensively described [2,3]. In the first steps of DNA replication, specific proteins are involved in origin recognition and unwinding of the DNA.…”

Section: Introductionmentioning

confidence: 99%

Nuclear recruitment of A l p 145 subunit of replication factor C in the early GI phase of the cell cycle in Faza 567 hepatoma cell line and hepatocyte primary cultures

et al. 1995

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Using a combination of immunoprecipitation and Western blotting with Faza 567 hepatoma cell extracts revealed that the large subunit of replication factor C (Alp145; mRFC140) was in a complex with proliferating cell nuclear antigen (PCNA). Western blotting showed that Alp145 was more abundant in nuclear extracts from butyrate-treated hepatoma cells which blocks the cells in the Cl phase of the cell cycle than from routinely cultured cells. Indirect immunoperoxidase analysis of Cl blocked Faza hepatoma cells localized Alp145 protein predominantly in the nucleoli. When hepatoma cells were stimulated to progress toward the S phase, Alp145 protein was then observed in both the cytoplasm and the nucleoplasm of these cells. Studies with early cultured normal hepatocytes which are progressing from Go towards G,, also showed a nucleolus distribution for Alp145. This is the first demonstration in mammalian cells that the large subunit of replication factor C is associated with PCNA in the nucleus and that its distribution within cells changes during the cell cycle.

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“…This residue is proposed to stack with the incoming dNTP [6]. (5) Conformational adaptation preceding the chemical stage of nucleotidyl transfer is a common step for many DNA polymerases and is thought to be responsible for polymerase fidelity [7,[26][27][28][29]. Tryptophan fluorescence quenching studies reveal that this stage is accompanied by conformational rearrangement of the protein which changes from the opened to the closed state [7,26].…”

Section: Our Modelmentioning

confidence: 99%

A model for DNA polymerase translocation: worm‐like movement of DNA within the binding cleft

1996

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Structure and function of the bacteriophage T4 DNA polymerase holoenzyme

Cited by 108 publications

References 116 publications

Replication Slippage of Different DNA Polymerases Is Inversely Related to Their Strand Displacement Efficiency

Replication Slippage of Different DNA Polymerases Is Inversely Related to Their Strand Displacement Efficiency

Nuclear recruitment of A l p 145 subunit of replication factor C in the early GI phase of the cell cycle in Faza 567 hepatoma cell line and hepatocyte primary cultures

A model for DNA polymerase translocation: worm‐like movement of DNA within the binding cleft

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