Mechanism of abasic lesion bypass catalyzed by a Y-family DNA polymerase

The 3 million-base pair genome of Sulfolobus solfataricus likely undergoes depurination/depyrimidination frequently in vivo. These unrepaired abasic lesions are expected to be bypassed by Dpo4, the only Y-family DNA polymerase from S. solfataricus. Interestingly, these error-prone Y-family enzymes have been shown to be physiologically vital in reducing the potentially negative consequences of DNA damage while paradoxically promoting carcinogenesis. Here we used Dpo4 as a model Y-family polymerase to establish the mechanistic basis for DNA lesion bypass. While showing efficient bypass, Dpo4 paused when incorporating nucleotides directly opposite and one position downstream from an abasic lesion because of a drop of several orders of magnitude in catalytic efficiency. Moreover, in disagreement with a previous structural report, Dpo4-catalyzed abasic bypass involves robust competition between the A-rule and the lesion loop-out mechanism and is governed by the local DNA sequence. Analysis of the strong pause sites revealed biphasic kinetics for incorporation indicating that Dpo4 primarily formed a nonproductive complex with DNA that converted slowly to a productive complex. These strong pause sites are mutational hot spots with the embedded lesion even affecting the efficiency of five to six downstream incorporations. Our results suggest that abasic lesion bypass requires tight regulation to maintain genomic stability.     

Structures of DNA polymerase beta with active-site mismatches suggest a transient abasic site intermediate during misincorporation

We report the crystallographic structures of DNA polymerase beta with dG-dAMPCPP and dC-dAMPCPP mismatches in the active site. These premutagenic structures were obtained with a nonhydrolyzable incoming nucleotide analog, dAMPCPP, and Mn(2+). Substituting Mn(2+) for Mg(2+) significantly decreases the fidelity of DNA synthesis. The structures reveal that the enzyme is in a closed conformation like that observed with a matched Watson-Crick base pair. The incorrect dAMPCPP binds in a conformation identical to that observed with the correct nucleotide. To accommodate the incorrect nucleotide and closed protein conformation, the template strand in the vicinity of the active site has shifted upstream over 3 A, removing the coding base from the active site and generating an abasic templating pocket. The primer terminus rotates as its complementary template base is repositioned. This rotation moves O3' of the primer terminus away from the alpha-phosphate of the incoming nucleotide, thereby deterring misincorporation.     

Sloppy bypass of an abasic lesion catalyzed by a Y-family DNA polymerase

DNA damage that eludes cellular repair pathways can arrest the replication machinery and stall the cell cycle. However, this damage can be bypassed by the Y-family DNA polymerases. Here, Dpo4, an archetypal Y-family member from the thermophilic Sulfolobus solfataricus, was used to extend our kinetic studies of the bypass of an abasic site, one of the most mutagenic and ubiquitous cellular lesions. A short oligonucleotide sequencing assay is developed to directly sequence DNA bypass products synthesized by Dpo4. Our results show that incorporation upstream of the abasic lesion is replicated error-free; yet dramatically, once Dpo4 encounters the lesion, synthesis became sloppy, with bypass products containing a myriad of mutagenic events. Incorporation of dAMP (29%) and dCMP (53%) opposite the abasic lesion at 37 degrees C correlates exceptionally well with our kinetic results and demonstrates two dominant bypass pathways via the A-rule and the lesion loop-out mechanism. Interestingly, the percentage of overall frameshift mutations increased from 71 (37 degrees C) to 87% (75 degrees C). Further analysis indicates that lesion bypass via the A-rule is strongly preferred over the lesion loop-out mechanism at higher temperatures and concomitantly reduces the occurrence of "-1 deletion" mutations observed opposite the lesion at lower temperatures. The bypass percentage via the latter pathway is confirmed by an enzymatic digestion assay, verifying the reliability of our sequencing assay. Our results demonstrate that an abasic lesion causes Dpo4 and possibly all Y-family members to switch from a normal to a very mutagenic mode of replication.     

Inefficient bypass of an abasic site by DNA polymerase eta

DNA polymerase eta (Pol eta) bypasses a cis-syn thymine-thymine dimer efficiently and accurately, and inactivation of Pol eta in humans results in the cancer-prone syndrome, the variant form of xeroderma pigmentosum. Also, Pol eta bypasses the 8-oxoguanine lesion efficiently by predominantly inserting a C opposite this lesion, and it bypasses the O(6)-methylguanine lesion by inserting a C or a T. To further assess the range of DNA lesions tolerated by Pol eta, here we examine the bypass of an abasic site, a prototypical noninstructional lesion. Steady-state kinetic analyses show that both yeast and human Pol eta are very inefficient in both inserting a nucleotide opposite an abasic site and in extending from the nucleotide inserted. Hence, Pol eta bypasses this lesion extremely poorly. These results suggest that Pol eta requires the presence of template bases opposite both the incoming nucleotide and the primer terminus to catalyze efficient nucleotide incorporation.     

Multiple solutions to inefficient lesion bypass by T7 DNA polymerase

We hypothesize that enzymatic switching during translesion synthesis (TLS) to relieve stalled replication forks occurs during transitions from preferential to disfavored use of damaged primer-templates, and that the polymerase or 3'-exonuclease used for each successive nucleotide incorporated is the one whose properties result in the highest efficiency and the highest fidelity of bypass. Testing this hypothesis requires quantitative determination of the relative lesion bypass ability of both TLS polymerases and major replicative polymerases. As a model of the latter, here we measure the efficiency and fidelity of cis-syn TT dimer and abasic site bypass using the structurally well-characterized T7 DNA polymerase. No bypass of either lesion occurred during a single round of synthesis, and the exonuclease activity of wild-type T7 DNA polymerase was critical in preventing TLS. When repetitive cycling of the exonuclease-deficient enzyme was allowed, limited bypass did occur but hundreds to thousands of cycles were required to achieve even a single bypass event. Analysis of TLS fidelity indicated that these rare bypass events involved rearrangements of the template and primer strands, insertions opposite the lesion, and combinations of these events, with the choice among these strongly depending on the sequence context of the lesion. Moreover, the presence of a lesion affected the fidelity of copying adjacent undamaged template bases, even when lesion bypass itself was correct. The results also indicate that a TT dimer presents a different type of block to the polymerase than an abasic site, even though both lesions are extremely potent blocks to processive synthesis. The approaches used here to quantify the efficiency and fidelity of TLS can be applied to other polymerase-lesion combinations, to provide guidance as to which of many possible polymerases is most likely to bypass various lesions in biological contexts.     

Role of DNA polymerase eta in the bypass of abasic sites in yeast cells

Abasic (AP) sites are major DNA lesions and are highly mutagenic. AP site-induced mutagenesis largely depends on translesion synthesis. We have examined the role of DNA polymerase η (Polη) in translesion synthesis of AP sites by replicating a plasmid containing a site-specific AP site in yeast cells. In wild-type cells, AP site bypass resulted in preferred C insertion (62%) over A insertion (21%), as well as −1 deletion (3%), and complex event (14%) containing multiple mutations. In cells lacking Polη (rad30), Rev1, Polζ (rev3), and both Polη and Polζ, translesion synthesis was reduced to 30%, 30%, 15% and 3% of the wild-type level, respectively. C insertion opposite the AP site was reduced in rad30 mutant cells and was abolished in cells lacking Rev1 or Polζ, but significant A insertion was still detected in these mutant cells. While purified yeast Polα effectively inserted an A opposite the AP site in vitro, purified yeast Polδ was much less effective in A insertion opposite the lesion due to its 3′→5′ proofreading exonuclease activity. Purified yeast Polη performed extension synthesis from the primer 3′ A opposite the lesion. These results show that Polη is involved in translesion synthesis of AP sites in yeast cells, and suggest that an important role of Polη is to catalyze extension following A insertion opposite the lesion. Consistent with these conclusions, rad30 mutant cells were sensitive to methyl methanesulfonate (MMS), and rev1 rad30 or rev3 rad30 double mutant cells were synergistically more sensitive to MMS than the respective single mutant strains.     

Role of a GAG hinge in the nucleotide-induced conformational change governing nucleotide specificity by T7 DNA polymerase

A nucleotide-induced change in DNA polymerase structure governs the kinetics of polymerization by high fidelity DNA polymerases. Mutation of a GAG hinge (G542A/G544A) in T7 DNA polymerase resulted in a 1000-fold slower rate of conformational change, which then limited the rate of correct nucleotide incorporation. Rates of misincorporation were comparable to that seen for wild-type enzyme so that the net effect of the mutation was a large decrease in fidelity. We demonstrate that a presumably modest change from glycine to alanine 20 Å from the active site can severely restrict the flexibility of the enzyme structure needed to recognize and incorporate correct substrates with high specificity. These results emphasize the importance of the substrate-induced conformational change in governing nucleotide selectivity by accelerating the incorporation of correct base pairs but not mismatches.     

In vitro selection of sequence contexts which enhance bypass of abasic sites and tetrahydrofuran by T4 DNA polymerase holoenzyme

The influence of sequence context on the ability of DNA polymerase to bypass sites of base loss was addressed using an in vitro selection system. Oligonucleotides containing either an aldehydic abasic site or tetrahydrofuran surrounded by four randomized bases on both the 5' and 3' sides were used as templates for synthesis by phage T4 DNA polymerase holoenzyme proficient or deficient in the 3'-->5' proofreading exonuclease activity. Successful bypass products were purified, subcloned and the sequences of approximately 100 subclones were determined for each of the four polymerase/lesion combinations tested. Between 7 and 19 % of the bypass products contained deletions of one to three nucleotides in the randomized region. In bypass products not containing deletions, biases for and against certain nucleotides were readily noticeable across the entire randomized region. Template strands from successful bypass products of abasic sites had a high frequency of T in most of the randomized positions, while those from bypass products of tetrahydrofuran had a high frequency of G at the positions immediately to the 3' and 5' side of the lesion. Consensus sequences were shared by successful bypass products of the same lesion but not between bypass products of the two lesions. The consensus sequence for efficient bypass of tetrahydrofuran was over-represented in several frames relative to the lesion. T4 DNA polymerase inserted A opposite abasic sites 63 % of the time in the presence of proofreading and 79 % of the time in its absence, followed by G>T>C, while the insertion of A opposite tetrahydrofuran ranged between 93 % and 100 % in the presence and absence of proofreading, respectively. Finally, sequence context influenced the choice of nucleotide inserted opposite abasic sites and consensus sequences which favored the incorporation of nucleotides other than A were defined.     

Functional evidence for a small and rigid active site in a high fidelity DNA polymerase: probing T7 DNA polymerase with variably sized base pairs

Hypotheses on the origins of high fidelity in replicative DNA polymerases have recently focused on the importance of geometric or steric effects in this selectivity. Here we reported a systematic study of the effects of base pair size in T7 DNA polymerase (pol), the replicative enzyme for bacteriophage T7. We varied base pair size in very small (0.25 A) increments by use of a series of nonpolar thymidine shape mimics having gradually increasing size. Steady-state kinetics were evaluated for the 5A7A exonuclease-deficient mutant in a 1:1 complex with thioredoxin. For T7 pol, we studied insertion of natural nucleotides opposite variably sized T analogs in the template and, conversely, for variably sized dTTP analogs opposite natural template bases. The enzyme displayed extremely high selectivity for a specific base pair size, with drops in efficiency of as much as 280-fold for increases of 0.4 A beyond an optimum size approximating the size of a natural pair. The enzyme also strongly rejected pairs that were smaller than the optimum by as little as 0.3 A. The size preferences with T7 DNA pol were generally smaller, and the steric rejection was greater than DNA pol I Klenow fragment, correlating with the higher fidelity of the former. The hypothetical effects of varied active site size and rigidity are discussed. The data lend direct support to the concept that active site tightness is a chief determinant of high fidelity of replicative polymerases and that a less rigid (looser) and larger active site can lead to lower fidelity.     

Stimulation of T7 DNA polymerase by a new phage-coded protein

Summary A bacteriophage-induced DNA-binding protein was purified from T7 infected E. coli. The protein has a molecular weight of about 25000, as judged by SDS-polyacrylamide gel electrophoresis. The purified protein binds to single-stranded but not to native T7 DNA. Like the T4 gene-32 protein and the 22000-dalton unwinding protein of E. coli, the T7 25000 protein lowers the melting temperature of poly d(A-T). Using partially single-stranded T7 DNA as template-primer, the protein stimulates in vitro DNA synthesis by T7 DNA polymerase about five-fold. It was also found that the DNA-unwinding protein of E. coli stimulates T7 DNA polymerase to approximately the same extent. However, neither of the unwinding proteins stimulate DNA polymerase I of E. coli.     

The abasic site as a challenge to DNA polymerase. A nuclear magnetic resonance study of G, C and T opposite a model abasic site

An abasic site in DNA creates a strong block to DNA polymerase and is a mutagenic base lesion. In this study, we present structural and dynamic properties of duplex oligodeoxynucleotides containing G, C and T opposite a model abasic site studied by one and two-dimensional nuclear magnetic resonance spectroscopy. We have demonstrated that A opposite the abasic site was positioned within the helix as if paired with T, and that the A residue melted co-operatively with the surrounding helix. We report here that G opposite the abasic site is also observed to be predominantly intrahelical in a normal anti conformation at low temperature. With increasing temperature, the mobility of the G residue increases rapidly and apparently is in a "melted state" well before denaturation of the helix. At low temperature, two species are found for T opposite the abasic site; one, intrahelical, one extrahelical. These species are in slow exchange with one another on a proton nuclear magnetic resonance time-scale. The two species then move into fast exchange with increasing temperature and the proportion of the extra-helical form increases. When C is positioned opposite the abasic site, both the C residue and the abasic sugar are extrahelical, the helix collapses, and the adjacent G.C base-pairs stack over one another. On the basis of these observations, we propose a model that explains why the abasic site acts to block DNA replication. Further, we suggest an explanation for the observed polymerase preference for base selection at abasic sites.     

The choice of nucleotide inserted opposite abasic sites formed within chromosomal DNA reveals the polymerase activities participating in translesion DNA synthesis

Abasic sites in genomic DNA can be a significant source of mutagenesis in biological systems, including human cancers. Such mutagenesis requires translesion DNA synthesis (TLS) bypass of the abasic site by specialized DNA polymerases. The abasic site bypass specificity of TLS proteins had been studied by multiple means in vivo and in vitro, although the generality of the conclusions reached have been uncertain. Here, we introduce a set of yeast reporter strains for investigating the in vivo specificity of abasic site bypass at numerous random positions within chromosomal DNA. When shifted to 37 °C, these strains underwent telomere uncapping and resection that exposed reporter genes within a long 3′ ssDNA overhang. Human APOBEC3G cytosine deaminase was expressed to create uracils in ssDNA, which were excised by uracil-DNA N-glycosylase. During repair synthesis, error-prone TLS bypassed the resulting abasic sites. Because of APOBEC3G's strict motif specificity and the restriction of abasic site formation to only one DNA strand, this system provides complete information about the location of abasic sites that led to mutations. We recapitulated previous findings on the roles of REV1 and REV3. Further, we found that sequence context can strongly influence the relative frequency of A or C insertion. We also found that deletion of Pol32, a non-essential common subunit of Pols δ and ζ, resulted in residual low-frequency C insertion dependent on Rev1 catalysis. We summarize our results in a detailed model of the interplay between TLS components leading to error-prone bypass of abasic sites. Our results underscore the utility of this system for studying TLS bypass of many types of lesions within genomic DNA.     

Redmond Red as a redox probe for the DNA-mediated detection of abasic sites

Redmond Red, a fluoropore containing a redox-active phenoxazine core, has been explored as a new electrochemical probe for the detection of abasic sites in double-stranded DNA. The electrochemical behavior of Redmond Red-modified DNA at gold surfaces exhibits stable, quasi-reversible voltammetry with a midpoint potential centered around -50 mV versus NHE. Importantly, with Redmond Red positioned opposite an abasic site within the DNA duplex, the electrochemical response is significantly enhanced compared to Redmond Red positioned across from a base. Redmond Red, reporting only if well-stacked in the duplex, represents a sensitive probe to detect abasic sites electrochemically in a DNA-mediated reaction.     

N6-methyldeoxyadenosine 5'-triphosphate as a probe of the fidelity mechanisms of bacteriophage T4 DNA polymerase

The incorporation of m6dATP by T4 DNA polymerase has been investigated. Unlike Escherichia coli DNA polymerase I (Engel, J.D., and von Hippel, P.H. (1978) J. Biol. Chem, 253, 935-939), the T4 enzyme discriminates at the insertion step against the methylated triphosphate as compared to the normal substrate (dATP). The apparent Km values measured in two ways agree with the overall 7-fold discrimination measured in double label experiments. The apparent Vmax values measured for net DNA synthesis are the same, while those measured for nucleotide turnover show that the rate for m6dATP is 2-fold greater than for dATP itself. The T4 enzyme results are consistent with the generally held theory that fidelity at the insertion step of DNA polymerization is determined by the relative free energies of primer-enzyme-triphosphate ternary complexes formed by competing, alternative substrate dNTPs. These results are also consistent with the view that these free energies chiefly depend on formation of satisfactory hydrogen bonds between the bases of the template and triphosphate.