competition between Pol X family members in VDJ recombination and general double strand break repair

competition between Pol X family members in VDJ recombination and general double strand break repair

As predicted by the amino acid sequence, the purified protein coded by Schizosaccharomyces pombe SPAC2F7.06c is a DNA polymerase (SpPol4) whose biochemical properties resemble those of other X family (PolX) members. Thus, this new PolX is template dependent, polymerizes in a distributive manner, lacks a detectable 30!50 proofreading activity and its preferred substrates are small gaps with a 50 phosphate group. Similarly to Polm, SpPol4 can incorporate a ribonucleotide (rNTP) into a primer DNA. However, it is not responsible for the 1 2 rNTPs proposed to be present at the mating type locus and those necessary for mating type switching. Unlike Polm, SpPol4 lacks terminal deoxynucleotidyltransferase activity and realigns the primer terminus to alternative template bases only under certain sequence contexts and, therefore, it is less errorprone than Polm. Nonetheless, the biochemical properties of this gap filling DNA polymerase are suitable for a possible role of SpPol4 in non homologous end joining. Unexpectedly based on sequence analysis, SpPol4 has deoxyribose phosphate lyase activity like Polb and Poll, and unlike Polm, suggesting also a role of this enzyme in base excision repair. Therefore, SpPol4 is a unique enzyme whose enzymatic properties are hybrid of those described for mammalian Polb, Poll and Polm.

Differences in the substrate specificity of mammalian family X DNA polymerases are proposed to partly depend on a loop (loop 1) upstream of the polymerase active site. To examine if this is the case in DNA polymerase j (pol j), here we characterize a variant of the human polymerase in which nine residues of loop 1 are replaced with four residues from the equivalent position in pol b. Crystal structures of the mutant enzyme bound to gapped DNA with and without a correct dNTP reveal that the change in loop 1 does not affect the overall structure of the protein. Consistent with these structural data, the mutant enzyme has relatively normal catalytic efficiency for correct incorporation, and it efficiently participates in non homologous end joining of double strand DNA breaks. However, DNA junctions recovered from end joining reactions are more diverse than normal, and the mutant enzyme is substantially less accurate than wild type pol j in three different biochemical assays. Comparisons of the binary and ternary complex crystal structures of mutant and wild type pol j suggest that loop 1 modulates pol j’s fidelity by controlling dNTP induced movements of the template strand and the primer terminal 30 OH as the enzyme transitions from an inactive to an active conformation.

As predicted by the amino acid sequence, the purified protein coded by Schizosaccharomyces pombe SPAC2F7.06c is a DNA polymerase (SpPol4) whose biochemical properties resemble those of other X family (PolX) members. Thus, this new PolX is template dependent, polymerizes in a distributive manner, lacksadetectable30!50proofreadingactivity and its preferred substrates are small gaps with a 50 phosphate group. Similarly to Polm, SpPol4 can incorporate a ribonucleotide (rNTP) into a primer DNA. However, it is not responsible for the 1 2 rNTPs proposed to be present at the mating type locus and those necessary for mating type switching. Unlike Polm, SpPol4 lacks terminal deoxynucleotidyl transferase activity and realigns the primer terminus to alternative template bases only under certain sequence contexts and, therefore, it is less error prone than Polm. Nonetheless, the biochemical prop erties of this gap filling DNA polymerase are suitable for a possible role of SpPol4 in non homologous end joining. Unexpectedly based on sequence ana lysis, SpPol4 has deoxyribose phosphate lyase activ ity like Polb and Poll, and unlike Polm, suggesting also a role of this enzyme in base excision repair. Therefore, SpPol4 is a unique enzyme whose enzym atic properties are hybrid of those described for mammalian Polb, Poll and Polm.