Processive searching ability varies among members of the gap-filling DNA polymerase X family

Abstract

DNA repair proteins must locate rare damaged sites within the genome. DNA polymerase β (Pol β), a member of the DNA polymerase X family that is involved in base excision repair, uses a processive hopping search mechanism to locate substrates. This effectively enhances its search footprint on DNA, increasing the probability of locating damaged sites. Processive searching has been reported or proposed for many DNA-binding proteins, raising the question of how widespread or specific to certain enzymes the ability to perform this function is. To provide insight into this question, we compared the ability of three homologous DNA Pol X family members to perform a processive search for 1-nucleotide gaps in DNA using a previously developed biochemical assay. We found that at near-predicted physiological ionic strengths, the intramolecular searching ability of Pol β is at least 4-fold higher than that of Pol μ and ∼2-fold higher than that of Pol λ. Pol β also was able to perform intersegmental transfer with the intersegmental searching ability of Pol β being at least 6- and ∼2-fold higher than that of Pols μ and λ, respectively. Mutational analysis suggested that differences in the N-terminal domains of these polymerases are responsible for the varying degrees of searching competence. Of note, the differences in processive searching ability observed among the DNA Pol X family members correlated with their proposed biological functions in base excision repair and nonhomologous end joining.

The deoxyribose phosphate lyase of DNA polymerase β suppresses a processive DNA synthesis to prevent trinucleotide repeat instability

“…A recent study from the Wilson group has shown that pol β uses positively charged lysine residues within the lyase domain to processively search for DNA damage, i.e. a 1 nt gap through hopping and intersegmental transfer along DNA ( 28 , 29 ). Once a gapped DNA is encountered, the lyase domain makes specific interactions with the gap to allow the lesion recognition.…”

“…It has also been shown that the dRP lyase activity of pol β is much faster than its DNA synthesis activity ( 20 ). A recent study from the Wilson group has also demonstrated that pol β dRP lyase domain performs a processive search for a 1 nt gap ( 28 , 29 ). This indicates that pol β dRP lyase initially searches for the 1 nt gap with a dRP group and removes the dRP group leaving the 1 nt gap ( 22 , 23 , 30 ) that is subsequently filled in by pol β gap-filling DNA synthesis via its polymerase activity.…”

DNA scanning by base excision repair enzymes and implications for pathway coordination

“…This efficiency ( E ) is different than catalytic efficiency ( k cat / K M ), which is a second order rate constant that measures productive associations between enzyme and substrate. The efficiency of an enzyme can be determined by pulse-chase or partition assays (17,18,22,23). In these assays, a competent enzyme complex with substrate(s) bound is challenged with competitor DNA (trap).…”

“…Indeed, examples in the literature of DNA repair enzymes not performing processive searching are rare ( E. coli proteins RNAP (26) and FtsK (27); however, these studies could not rule out short scanning events (<115 bp). To provide insight into this question, we measured the processive searching abilities of the DNA polymerase X family members that are capable of catalyzing 1-nt gap filing DNA synthesis (Pols β, μ, and λ) (23).…”

“…However, recent studies support a mechanism of intersegmental transfer that involves a microscopically dissociated protein jumping to a neighboring DNA segment (44). Using a substrate that was previously utilized to characterize the intersegmental transfer ability of the DNA glycosylase AAG, we recently demonstrated that Pol β can perform intersegmental transfer (23,44). Considering the high concentration of DNA in the nucleus, intersegmental transfer may be a highly operational mode of facilitated diffusion inside the cell.…”