Biochemical Properties of Saccharomyces cerevisiae DNA Polymerase IV

Abstract

Although mammals encode multiple family X DNA polymerases implicated in DNA repair, Saccharomyces cerevisiae has only one, DNA polymerase IV (pol IV). To better understand the repair functions of pol IV, here we characterize its biochemical properties. Like mammalian pol ␤ and pol , but not pol , pol IV has intrinsic 5-2-deoxyribose-5-phosphate lyase activity. Pol IV has low processivity and can fill short gaps in DNA. Unlike the case with pol ␤ and pol , the gap-filling activity of pol IV is not enhanced by a 5-phosphate on the downstream primer but is stimulated by a 5-terminal synthetic abasic site. Pol IV incorporates rNTPs into DNA with an unusually high efficiency relative to dNTPs, a property in common with pol but not pol ␤ or pol . Finally, pol IV is highly inaccurate, with an unusual error specificity indicating the ability to extend primer termini with limited homology. These properties are consistent with a possible role for pol IV in base excision repair and with its known role in non-homologous end joining of double strand breaks, perhaps including those with damaged ends.Family X DNA polymerases are evolutionarily conserved, single-subunit enzymes devoid of 3Ј 3 5Ј exonuclease activity. The most extensively studied family X polymerase (pol) 1 is mammalian DNA pol ␤, which has a major role in base excision repair (1). Other mammalian family X polymerases also implicated in DNA repair include template-dependent pol (2) and pol (3-5) and template-independent terminal deoxyribonucleotidyl transferase (TdT) (6). These enzymes share an 8-kDa domain and a polymerase domain comprised of fingers, palm, and thumb subdomains. Nonetheless, they differ in several amino acids likely to be important for function. Pol , pol , and TdT also have N-terminal BRCT domains not present in pol ␤. Mammalian family X polymerases also have different biochemical properties. TdT adds nucleotides in a template-independent manner, consistent with its role in increasing the diversity of antigen receptor genes by adding nucleotides to the ends of coding segments during V(D)J recombination (7). Pol ␤ is template-dependent and moderately accurate for both substitution and single base deletion errors; it fills short gaps in DNA in a manner stimulated by a 5Ј-phosphate on the downstream primer (8) and has an intrinsic dRP lyase activity (9). These properties are all consistent with the well established role of pol ␤ in BER.Pol shares most of the above-mentioned properties with pol ␤, suggesting that pol may also function in BER (10, 11). Pol binds dNTPs with high affinity, consistent with polymerization when cellular dNTP concentrations are low, e.g. in cells not in S phase (12). Pol generates single base deletions, especially in non-iterated sequence contexts, at a much higher rate than pol ␤, indicating an ability to use template-primers with minimal base pairing homology. These properties, in combination with a BRCT domain and the ability to physically and functionally interact with several proteins involved in NHEJ of double st...

Evidence that base stacking potential in annealed 3′ overhangs determines polymerase utilization in yeast nonhomologous end joining

“…We therefore asked whether the Pol4 lyase can participate in 5' dRP processing during NHEJ ( Figure 1B). pol4-KK247∷248RR mutation eliminates two lysines predicted to catalyze the lyase reaction based on alignments with Pol β, although this mutation does not completely disable the lyase activity in vitro [17]. As previously observed [9], pol4-KK247∷248RR cells showed a mild defect at rejoining a DSB containing simple Pol4-dependent gaps ( Figure 3A, first joint).…”

“…This interpretation is supported by in vitro analyses of Pol4 and the related mammalian Pol X NHEJ polymerases, Pol μ and Pol λ, including extensive structural data, which have revealed several unique features of the Pol X polymerases [17,24,[29][30][31][32][33][34]. For example, the Pol X NHEJ polymerases make fewer template strand contacts [24] and have a propensity toward strand slippage [17,30,31], mismatch extension [32] and lesion bypass [33], observations consistent with a reduced dependence on a stable primer-template pairing. Indeed, a "gradient" of polymerase properties has been described in which Pol μ has the remarkable ability to catalyze template-dependent synthesis without any initial primer-template pairing [34].…”

“…The other issue addressed in this report is an explicit test of the short patch/long patch model of 5' processing in NHEJ (Figure 1), suggested by the presence of 5' dRP lyase activity in Pol4 [17] and the previously proposed role of Rad27 in NHEJ [20,21]. We were unable to provide clear support for either model, since ends with 5' dRP termini could be rejoined in the absence of both the lyase activity of Pol4 and Rad27 (Figure 3).…”

“…However, the minor defect in pol4-KK247∷248RR cells could also be due to residual Pol4 lyase activity as observed in vitro [17]. As an alternate way to address 5' dRP removal, we constructed a DSB with 5' dRP termini that lacks gaps.…”

“…In the long patch pathway, either Pol δ or Pol β performs displacement synthesis [14,15], yielding a 5' flap, which is cleaved by FEN-1 (Rad27 in yeast) [16]. Compellingly, Pol4 and Pol β are each nonprocessive gap-filling polymerases, and both have 5' dRP lyase activity [17][18][19]. Also, Rad27 physically interacts with Dnl4 and Pol4, can process 5' flaps at DSBs in vitro [20], and has been implicated in NHEJ of ends with 5' flaps in vivo [21].…”