DNA polymerase δ-interacting protein 2 is a processivity factor for DNA polymerase λ during 8-oxo-7,8-dihydroguanine bypass.

The bypass of DNA lesions by the replication fork requires a switch between the replicative DNA polymerase (Pol) and a more specialized translesion synthesis (TLS) Pol to overcome the obstacle. DNA Pol δ-interacting protein 2 (PolDIP2) has been found to physically interact with Pol η, Pol ζ, and Rev1, suggesting a possible role of PolDIP2 in the TLS reaction. However, the consequences of PolDIP2 interaction on the properties of TLS Pols remain unknown. Here, we analyzed the effects of PolDIP2 on normal and TLS by five different human specialized Pols from three families: Pol δ (family B), Pol η and Pol ι (family Y), and Pol λ and Pol ß (family X). Our results show that PolDIP2 also physically interacts with Pol λ, which is involved in the correct bypass of 8-oxo-7,8-dihydroguanine (8-oxo-G) lesions. This interaction increases both the processivity and catalytic efficiency of the error-free bypass of a 8-oxo-G lesion by both Pols η and λ, but not by Pols ß or ι. Additionally, we provide evidence that PolDIP2 stimulates Pol δ without affecting its fidelity, facilitating the switch from Pol δ to Pol λ during 8-oxo-G TLS. PolDIP2 stimulates Pols λ and η mediated bypass of other common DNA lesions, such as abasic sites and cyclobutane thymine dimers. Finally, PolDIP2 silencing increases cell sensitivity to oxidative stress and its effect is further potentiated in a Pol λ deficient background, suggesting that PolDIP2 is an important mediator for TLS.

A switch between DNA polymerases δ and λ promotes error-free bypass of 8-oxo-G lesions.

7,8-Dihydro-8-oxoguanine (8-oxo-G) is a highly abundant and mutagenic lesion. Replicative DNA polymerases (pols) are slowed down at 8-oxo-G and insert both correct cytosine (C) and incorrect adenine (A) opposite 8-oxo-G, but they preferentially extend A:8-oxo-G mispairs. Nevertheless, 8-oxo-G bypass is fairly accurate in vivo. Thus, the question how correct bypass of 8-oxo-G lesions is accomplished despite the poor extension of C:8-oxo-G base pairs by replicative pols remains unanswered. Here we show that replicative pol δ pauses in front of 8-oxo-G and displays difficulties extending from correct C:8-oxo-G in contrast to extension from incorrect A:8-oxo-G. This leads to stalling of pol δ at 8-oxo-G after incorporation of correct C. This stalling at C:8-oxo-G can be overcome by a switch from pol δ to pols λ, ß, or η, all of which are able to assist pol δ in 8-oxo-G bypass by translesion synthesis (TLS). Importantly, however, only pol λ selectively catalyzes the correct TLS past 8-oxo-G, whereas pols ß and η show no selectivity and even preferentially enhance incorrect TLS. The selectivity of pol λ to promote the correct bypass depends on its N-terminal domain. Furthermore, pol λ(-/-) mouse embryonic fibroblast extracts display reduced 8-oxo-G TLS. Finally, the correct bypass of 8-oxo-G in gapped plasmids in mouse embryonic fibroblasts and HeLa cells is promoted in the presence of pol λ. Our findings suggest that even though 8-oxo-G is not a blocking lesion per se, correct replication over 8-oxo-G is promoted by a pol switch between pols δ and λ.

Characterization of the 6-methyl isoxanthopterin (6-MI) base analog dimer, a spectroscopic probe for monitoring guanine base conformations at specific sites in nucleic acids.

We here characterize local conformations of site-specifically placed pairs of guanine (G) residues in RNA and DNA, using 6-methyl isoxanthopterin (6-MI) as a conformational probe. 6-MI is a base analog of G and spectroscopic signals obtained from pairs of adjacent 6-MI residues reflect base-base interactions that are sensitive to the sequence context, local DNA conformation and solvent environment of the probe bases. CD signals show strong exciton coupling between stacked 6-MI bases in double-stranded (ds) DNA; this coupling is reduced in single-stranded (ss) DNA sequences. Solvent interactions reduce the fluorescence of the dimer probe more efficiently in ssDNA than dsDNA, while self-quenching between 6-MI bases is enhanced in dsDNA. 6-MI dimer probes closely resemble adjacent GG residues, in that these probes have minimal effects on the stability of dsDNA and on interactions with solvent additive betaine. They also serve as effective template bases, although further polymerase-dependent extension of DNA primers past 6-MI template bases is significantly inhibited. These probes are also used to monitor DNA 'breathing' at model replication forks. The 6-MI dimer probe can serve in many contexts as a useful tool to investigate GG conformations at specific sites within the nucleic acid frameworks of functioning macromolecular machines in solution.

8-oxo-guanine bypass by human DNA polymerases in the presence of auxiliary proteins.

Specialized DNA polymerases (DNA pols) are required for lesion bypass in human cells. Auxiliary factors have an important, but so far poorly understood, role. Here we analyse the effects of human proliferating cell nuclear antigen (PCNA) and replication protein A (RP-A) on six different human DNA pols--belonging to the B, Y and X classes--during in vitro bypass of different lesions. The mutagenic lesion 8-oxo-guanine (8-oxo-G) has high miscoding potential. A major and specific effect was found for 8-oxo-G bypass with DNA pols lambda and eta. PCNA and RP-A allowed correct incorporation of dCTP opposite a 8-oxo-G template 1,200-fold more efficiently than the incorrect dATP by DNA pol lambda, and 68-fold by DNA pol eta, respectively. Experiments with DNA-pol-lambda-null cell extracts suggested an important role for DNA pol lambda. On the other hand, DNA pol iota, together with DNA pols alpha, delta and beta, showed a much lower correct bypass efficiency. Our findings show the existence of an accurate mechanism to reduce the deleterious consequences of oxidative damage and, in addition, point to an important role for PCNA and RP-A in determining a functional hierarchy among different DNA pols in lesion bypass.

In vitro gap-directed translesion DNA synthesis of an abasic site involving human DNA polymerases epsilon, lambda, and beta.

DNA polymerase (pol) ε is thought to be the leading strand replicase in eukaryotes, whereas pols λ and ß are believed to be mainly involved in re-synthesis steps of DNA repair. DNA elongation by the human pol ε is halted by an abasic site (apurinic/apyrimidinic (AP) site). In this study, we present in vitro evidence that human pols λ, ß, and η can perform translesion synthesis (TLS) of an AP site in the presence of pol ε, likely by initiating the 3'OHs created at the lesion by the arrested pol ε. However, in the case of pols λ and ß, this TLS requires the presence of a DNA gap downstream from the product synthesized by the pol ε, and the optimal gap for efficient TLS is different for the two polymerases. The presence of gaps did not affect the TLS capacity of human pol η. Characterization of the reaction products showed that pol ß inserted dAMP opposite the AP site, whereas gap filling synthesis by pol λ resulted in single or double deletions opposite the lesion. The synthesis up to the AP site by pol ε and the subsequent TLS by pols λ and ß are not influenced by human processivity factor proliferating cell nuclear antigen and human single-stranded DNA-binding protein replication protein A. The bypass capacity of pol λ at the AP site is greatly reduced when a truncated form of the enzyme, which has lost the BRCA1 C-terminal and proline-rich domains, is used. Collectively, our in vitro results support the existence of a mechanism of gap-directed TLS at an AP site involving a switch between the replicative pol ε and the repair pols λ and ß.

Effect of 8-oxoguanine and abasic site DNA lesions on in vitro elongation by human DNA polymerase in the presence of replication protein A and proliferating-cell nuclear antigen.

DNA pol (polymerase) is thought to be the leading strand replicase in eukaryotes. In the present paper, we show that human DNA pol can efficiently bypass an 8-oxo-G (7,8-dihydro-8-oxoguanine) lesion on the template strand by inserting either dCMP or dAMP opposite to it, but it cannot bypass an abasic site. During replication, DNA pols associate with accessory proteins that may alter their bypass ability. We investigated the role of the human DNA sliding clamp PCNA (proliferating-cell nuclear antigen) and of the human single-stranded DNA-binding protein RPA (replication protein A) in the modulation of the DNA synthesis and translesion capacity of DNA pol . RPA inhibited the elongation by human DNA pol on templates annealed to short primers. PCNA did not influence the elongation by DNA pol and had no effect on inhibition of elongation caused by RPA. RPA inhibition was considerably reduced when the length of the primers was increased. On templates bearing the 8-oxo-G lesion, this inhibitory effect was more pronounced on DNA replication beyond the lesion, suggesting that RPA may prevent extension by DNA pol after incorporation opposite an 8-oxo-G. Neither PCNA nor RPA had any effect on the inability of DNA pol to replicate past the AP site, independent of the primer length.

Replication protein A and proliferating cell nuclear antigen coordinate DNA polymerase selection in 8-oxo-guanine repair.

The adenine misincorporated by replicative DNA polymerases (pols) opposite 7,8-dihydro-8-oxoguanine (8-oxo-G) is removed by a specific glycosylase, leaving the lesion on the DNA. Subsequent incorporation of C opposite 8-oxo-G on the resulting 1-nt gapped DNA is essential for the removal of the 8-oxo-G to prevent G-C to T-A transversion mutations. By using model DNA templates, purified DNA pols beta and lambda and knockout cell extracts, we show here that the auxiliary proteins replication protein A and proliferating cell nuclear antigen act as molecular switches to activate the DNA pol lambda- dependent highly efficient and faithful repair of A:8-oxo-G mismatches in human cells and to repress DNA pol beta activity. By using an immortalized human fibroblast cell line that has the potential to induce cancer in mice, we show that the development of a tumoral phenotype in these cells correlated with a differential expression of DNA pols lambda and beta.

Intrinsic properties of the two replicative DNA polymerases of Pyrococcus abyssi in replicating abasic sites: possible role in DNA damage tolerance?

Spontaneous and induced abasic sites in hyperthermophiles DNA have long been suspected to occur at high frequency. Here, Pyrococcus abyssi was used as an attractive model to analyse the impact of such lesions onto the maintenance of genome integrity. We demonstrated that endogenous AP sites persist at a slightly higher level in P. abyssi genome compared with Escherichia coli. Then, the two replicative DNA polymerases, PabpolB and PabpolD, were characterized in presence of DNA containing abasic sites. Both Pabpols had abortive DNA synthesis upon encountering AP sites. Under running start conditions, PabpolB could incorporate in front of the damage and even replicate to the full-length oligonucleotides containing a specific AP site, but only when present at a molar excess. Conversely, bypassing activity of PabpolD was strictly inhibited. The tight regulation of nucleotide incorporation opposite the AP site was assigned to the efficiency of the proof-reading function, because exonuclease-deficient enzymes exhibited effective TLS. Steady-state kinetics reinforced that Pabpols are high-fidelity DNA polymerases onto undamaged DNA. Moreover, Pabpols preferentially inserted dAMP opposite an AP site, albeit inefficiently. While the template sequence of the oligonucleotides did not influence the nucleotide insertion, the DNA topology could impact on the progression of Pabpols. Our results are interpreted in terms of DNA damage tolerance.

The checkpoint clamp, Rad9-Rad1-Hus1 complex, preferentially stimulates the activity of apurinic/apyrimidinic endonuclease 1 and DNA polymerase beta in long patch base excision repair.

Growing evidence suggests that the Rad9-Rad1-Hus1 complex (the 9-1-1 complex), besides its functions in DNA damage sensing and signaling pathways, plays also a direct role in various DNA repair processes. Recent studies have demonstrated that the 9-1-1 complex physically and functionally interacts with several components of the base excision repair (BER) machinery namely DNA polymerase beta (Pol beta), flap endonuclease 1 (Fen 1), DNA ligase I (Lig I) and the MutY homologue of Schizosaccharomyces pombe. In this work, we found for the first time that the 9-1-1 complex interacts in vitro and in vivo with the apurinic/apyrimidinic endonuclease 1 (APE 1), an early component of BER, and can stimulate its AP-endonuclease activity. Moreover, we show that the 9-1-1 complex possesses a stimulatory effect on long patch base excision repair (LP-BER) reconstituted in vitro. The enhancement of LP-BER activity is due to the specific stimulation of the two early components of the repair machinery, namely APE 1 and Pol beta, suggesting a hierarchy of interactions between the 9-1-1 complex and the BER proteins acting in the repairosome. Overall, our results indicate that the 9-1-1 complex is directly involved in LP-BER, thus providing a possible link between DNA damage checkpoints and BER.

Expanding the repertoire of DNA polymerase substrates: template-instructed incorporation of non-nucleoside triphosphate analogues by DNA polymerases beta and lambda.

We have recently shown that neither the base nor the sugar moieties of a nucleotide is an essential feature for its incorporation by DNA polymerases (pols) lambda and beta. Here we present the identification of novel non-nucleoside triphosphate (NNTP) derivatives belonging to three classes: (i) non-substrate-specific inhibitors of DNA pol lambda; (ii) substrate inhibitors which could preferentially be incorporated by either DNA pol lambda wild type or its Y505A mutant and (iii) the substrate inhibitor N-(Biphenylcarbonyl)-4-oxobutyl triphosphate which could be incorporated exclusively by DNA pol beta in a Mg2+-dependent manner, and preferentially pairs with A on the template. This compound represents the first example of a substrate lacking both nucleobase and ribose residue, showing distinct base-pairing properties with normal bases. Therefore, this NNTP analog can be considered as the prototype of an entirely novel class of DNA pol substrates.

The balance between the rates of incorporation and pyrophosphorolytic removal influences the HIV-1 reverse transcriptase bypass of an abasic site with deoxy-, dideoxy-, and ribonucleotides.

Abasic (AP) sites pose a potential danger to HIV-1 replication. HIV-1 RT has been shown to preferentially incorporate purines opposite an AP site, and subsequently extend from the lesion. While it is clear that AP sites are bypassed inefficiently and are major sites of RT pausing, detailed kinetic analysis of the relative contributions of both the incorporation and the pyrophosphorolytic reactions in translesion synthesis by HIV-RT is still lacking. Investigation of the molecular basis of these processes is important, in light of the fact that HIV-1 RT is the major target for anti-HIV chemotherapy, and its low fidelity is an essential determinant of the extraordinary genetic variability of HIV-1, which is important for the appearance of mutant viruses resistant to chemotherapy. Here, we analyzed the effects of the presence of an AP site on the template strand on the catalytic properties of the DNA-dependent polymerization reaction as well as on the phosphorolytic activity of HIV-1 RT, in the presence of deoxy-, dideoxy,- and ribonucleotides. We find that AP sites can substantially influence the substrate specificity of HIV-1 RT and that pyrophosphorolysis plays a significant role in determining the ability of HIV-1 RT to (mis)incorporate nucleotides.

Accessory proteins assist exonuclease-deficient bacteriophage T4 DNA polymerase in replicating past an abasic site.

Replicative DNA polymerases, such as T4 polymerase, possess both elongation and 3'-5' exonuclease proofreading catalytic activities. They arrest at the base preceding DNA damage on the coding DNA strand and specialized DNA polymerases have evolved to replicate across the lesion by a process known as TLS (translesion DNA synthesis). TLS is considered to take place in two steps that often require different enzymes, insertion of a nucleotide opposite the damaged template base followed by extension from the inserted nucleotide. We and others have observed that inactivation of the 3'-5' exonuclease function of T4 polymerase enables TLS across a single site-specific abasic [AP (apurinic/apyrimidinic)] lesion. In the present study we report a role for auxiliary replicative factors in this reaction. When replication is performed with a large excess of DNA template over DNA polymerase in the absence of auxiliary factors, the exo- polymerase (T4 DNA polymerase deficient in the 3'-5' exonuclease activity) inserts one nucleotide opposite the AP site but does not extend past the lesion. Addition of the clamp processivity factor and the clamp loader complex restores primer extension across an AP lesion on a circular AP-containing DNA substrate by the exo- polymerase, but has no effect on the wild-type enzyme. Hence T4 DNA polymerase exhibits a variety of responses to DNA damage. It can behave as a replicative polymerase or (in the absence of proofreading activity) as a specialized DNA polymerase and carry out TLS. As a specialized polymerase it can function either as an inserter or (with the help of accessory proteins) as an extender. The capacity to separate these distinct functions in a single DNA polymerase provides insight into the biochemical requirements for translesion DNA synthesis.

Human replication protein A can suppress the intrinsic in vitro mutator phenotype of human DNA polymerase lambda.

DNA polymerase lambda (pol lambda) is a member of the X family DNA polymerases and is endowed with multiple enzymatic activities. In this work we investigated the in vitro miscoding properties of full-length, human pol lambda either in the absence or in the presence of the human auxiliary proteins proliferating cell nuclear antigen (PCNA) and replication protein A (RP-A). Our data suggested that (i) pol lambda had an intrinsic ability to create mismatches and to incorporate ribonucleotides at nearly physiological Mn++ and Mg++ concentrations; (ii) the sequence of the template-primer could influence the misincorporation frequency of pol lambda; (iii) pol lambda preferentially generated G:T and G:G mismatches; (iv) RP-A, but not PCNA, selectively prevented misincorporation of an incorrect nucleotide by pol lambda, without affecting correct incorporation and (v) this inhibitory effect required a precise ratio between the concentrations of pol lambda and RP-A. Possible physiological implications of these findings for the in vivo fidelity of pol lambda are discussed.

Incorporation of non-nucleoside triphosphate analogues opposite to an abasic site by human DNA polymerases beta and lambda.

A novel class of non-nucleoside triphosphate analogues, bearing hydrophobic groups sterically similar to nucleosides linked to the alpha-phosphate but lacking the chemical functional groups of nucleic acids, were tested against six different DNA polymerases (polymerases). Human polymerases alpha, beta and lambda, and Saccharomyces cerevisiae polymerase IV, were inhibited with different potencies by these analogues. On the contrary, Escherichia coli polymerase I and HIV-1 reverse transcriptase were not. Polymerase beta incorporated these derivatives in a strictly Mn++-dependent manner. On the other hand, polymerase lambda could incorporate some alkyltriphosphate derivatives with both Mg++ and Mn++, but only opposite to an abasic site on the template strand. The active site mutant polymerase lambda Y505A showed an increased ability to incorporate the analogues. These results show for the first time that neither the base nor the sugar moieties of nucleotides are required for incorporation by family X DNA polymerases.

Mutagenesis of human DNA polymerase lambda: essential roles of Tyr505 and Phe506 for both DNA polymerase and terminal transferase activities.

DNA polymerase (pol) lambda is homologous to pol beta and has intrinsic polymerase and terminal transferase activities. However, nothing is known about the amino acid residues involved in these activities. In order to precisely define the nucleotide-binding site of human pol lambda, we have mutagenised two amino acids, Tyr505 and the neighbouring Phe506, which were predicted by structural homology modelling to correspond to the Tyr271 and Phe272 residues of pol beta, which are involved in nucleotide binding. Our analysis demonstrated that pol lambda Phe506Arg/Gly mutants possess very low polymerase and terminal transferase activities as well as greatly reduced abilities for processive DNA synthesis and for carrying on translesion synthesis past an abasic site. The Tyr505Ala mutant, on the other hand, showed an altered nucleotide binding selectivity to perform the terminal transferase activity. Our results suggest the existence of a common nucleotide-binding site for the polymerase and terminal transferase activities of pol lambda, as well as distinct roles of the amino acids Tyr505 and Phe506 in these two catalytic functions.

Effect of manganese on in vitro replication of damaged DNA catalyzed by the herpes simplex virus type-1 DNA polymerase.

In vitro bypass of damaged DNA by replicative DNA polymerases is usually blocked by helix-distorting or bulky DNA lesions. In this study, we report that substitution of the divalent metal ion Mg2+ with Mn2+ promotes quantitative replication of model DNA substrates containing the major cisplatin or N-2-acetylaminofluorene adducts by the catalytic subunit (UL30) of the replicative DNA polymerase of herpes simplex virus. The ability of Mn2+ ions to confer bypass of bulky lesions was not observed with other replicative DNA polymerases of the B family, such as bacteriophage T4 or delta polymerases. However, for these enzymes, manganese induced the incorporation of one nucleotide opposite the first (3') guanine of the d(GpG) intrastrand cisplatin lesion. Translesion replication of the cisplatin adduct by UL30 led to the incorporation of mismatched bases, with the preferential incorporation of dAMP opposite the 3' guanine of the lesion. Furthermore, substitution of MgCl2 with MnCl2 greatly inhibited the 3' to 5' exonuclease of UL30 but had a far lesser effect on that of T4 DNA polymerase. Finally, manganese induced a conformational change in the structure of UL30 bound to the platinated substrate. Taken together, the latter findings suggest a mechanism by which manganese might allow UL30 to efficiently promote translesion DNA synthesis in vitro.

The human Rad9/Rad1/Hus1 damage sensor clamp interacts with DNA polymerase beta and increases its DNA substrate utilisation efficiency: implications for DNA repair.

In eukaryotic cells, checkpoints are activated in response to DNA damage. This requires the action of DNA damage sensors such as the Rad family proteins. The three human proteins Rad9, Rad1 and Hus1 form a heterotrimeric complex (called the 9-1-1 complex) that is recruited onto DNA upon damage. DNA damage also triggers the recruitment of DNA repair proteins at the lesion, including specialized DNA polymerases. In this work, we showed that the 9-1-1 complex can physically interact with DNA polymerase beta in vitro. Functional analysis revealed that the 9-1-1 complex had a stimulatory effect on DNA polymerase beta activity. However, the presence of 9-1-1 complex neither affected DNA polymerase lambda, another X family DNA polymerase, nor the two replicative DNA polymerases alpha and delta. DNA polymerase beta stimulation resulted from an increase in its affinity for the primer-template and the interaction with the 9-1-1 complex stimulated deoxyribonucleotides misincorporation by DNA polymerase beta. In addition, the 9-1-1 complex enhanced DNA strand displacement synthesis by DNA polymerase beta on a 1 nt gap DNA substrate. Our data raise the possibility that the 9-1-1 complex might attract DNA polymerase beta to DNA damage sites, thus connecting directly checkpoints and DNA repair.

Herpes simplex virus type-1: a model for genome transactions.

In many respects, HSV-1 is the prototypic herpes virus. However, HSV-1 also serves as an excellent model system to study genome transactions, including DNA replication, homologous recombination, and the interaction of DNA replication enzymes with DNA damage. Like eukaryotic chromosomes, the HSV-1 genome contains multiple origins of replication. Replication of the HSV-1 genome is mediated by the concerted action of several virus-encoded proteins that are thought to assemble into a multiprotein complex. Several host-encoded factors have also been implicated in viral DNA replication. Furthermore, replication of the HSV-1 genome is known to be closely associated with homologous recombination that, like in many cellular organisms, may function in recombinational repair. Finally, recent data have shed some light on the interaction of essential HSV-1 replication proteins, specifically its DNA polymerase and DNA helicases, with damaged DNA.

On the role of proofreading exonuclease in bypass of a 1,2 d(GpG) cisplatin adduct by the herpes simplex virus-1 DNA polymerase.

UL30, the herpes simplex virus type-1 DNA polymerase, stalls at the base preceding a cisplatin crosslinked 1,2 d(GpG) dinucleotide and engages in a futile cycle of incorporation and excision by virtue of its 3'-5' exonuclease. Therefore, we examined the translesion synthesis (TLS) potential of an exonuclease-deficient UL30 (UL30D368A). We found that UL30D368A did not perform complete translesion synthesis but incorporated one nucleotide opposite the first base of the adduct. This addition was affected by the propensity of the enzyme to dissociate from the damaged template. Consequently, addition of the polymerase processivity factor, UL42, increased nucleotide incorporation opposite the lesion. The addition of Mn(2+), which was previously shown to support translesion synthesis by wild-type UL30, also enabled limited bypass of the adduct by UL30D368A. We show that the primer terminus opposite the crosslinked d(GpG) dinucleotide and at least three bases downstream of the lesion is unpaired and not extended by the enzyme. These data indicate that the primer terminus opposite the lesion may be sequestered into the exonuclease site of the enzyme. Consequently, elimination of exonuclease activity alone, without disrupting binding, is insufficient to permit bypass of a bulky lesion by this enzyme.

Inactivation of the 3'-5' exonuclease of the replicative T4 DNA polymerase allows translesion DNA synthesis at an abasic site.

Here, we have investigated the consequences of the loss of proof-reading exonuclease function on the ability of the replicative T4 DNA polymerase (gp43) to elongate past a single abasic site located on model DNA substrates. Our results show that wild-type T4 DNA polymerase stopped at the base preceding the lesion on two linear substrates having different sequences, whereas the gp43 D219A exonuclease-deficient mutant was capable of efficient bypass when replicating the same substrates. The structure of the DNA template did not influence the behavior of the exonuclease-proficient or deficient T4 DNA polymerases. In fact, when replicating a damaged "minicircle" DNA substrate constructed by circularizing one of the linear DNA, elongation by wild-type enzyme was still completely blocked by the abasic site, while the D219A mutant was capable of bypass. During DNA replication, the T4 DNA polymerase associates with accessory factors whose combined action increases the polymerase-binding capacity and processivity, and could modulate the behavior of the enzyme towards an abasic site. We thus performed experiments measuring the ability of wild-type and exonuclease-deficient T4 DNA polymerases, in conjunction with these replicative accessory proteins, to perform translesion DNA replication on linear or circular damaged DNA substrates. We found no evidence of either stimulation or inhibition of the bypass activities of the wild-type and exonuclease-deficient forms of T4 DNA polymerase following addition of the accessory factors, indicating that the presence or absence of the proof-reading activity is the major determinant in dictating translesion synthesis of an abasic site by T4 DNA polymerase.