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...