Evidence for an Imino Intermediate in the DNA Polymerase β Deoxyribose Phosphate Excision Reaction

Abstract

A recent study demonstrated that rat DNA polymerase ␤ (␤-pol) releases 5 -deoxyribose phosphate (dRP) termini from preincised apurinic/apyrimidinic DNA, a substrate generated during certain types of base excision repair. This catalytic activity resides within the aminoterminal, 8-kDa domain of ␤-pol and occurs via ␤-elimination as opposed to hydrolysis (Matsumoto, Y., and Kim, K. (1995) Science 269, 699 -702). The latter finding suggested that the dRP excision reaction might proceed via an imine intermediate. In order to test this hypothesis, we attempted to trap ␤-pol on preincised apurinic/ apyrimidinic DNA using NaBH 4 as the reducing agent. Both 8-kDa domain-DNA and intact ␤-pol-DNA complexes were detected and identified by autoradiography coupled to immunoblotting. Our results indicate that the chemical mechanism of the ␤-pol dRpase reaction does proceed through an imine enzyme-DNA intermediate and that the active site residue responsible for dRP release must therefore contain a primary amine.Vertebrate DNA polymerase ␤ (␤-pol), 1 a constitutively-expressed monomeric protein ranging from 39 to 45 kDa, has been implicated in DNA repair. Specifically, in vitro experiments indicate that ␤-pol can perform two of the five reactions involved in base excision repair, excision of a 5Ј-terminal dRP from a preincised AP site (1) and DNA synthesis to fill the single-nucleotide gap (2-5). The overall pathway for base excision repair that has been initiated by simple N-glycosylases (glycosylases lacking a concomitant AP lyase activity) is believed to proceed according to the following scheme. (i) A damage-specific DNA N-glycosylase creates an AP site by cleaving an N-C glycosyl bond, thereby releasing a modified base; (ii) a class II AP endonuclease incises the DNA 5Ј to the AP site; (iii) ␤-pol or another dRpase excises the 5Ј dRP terminus to leave a single-nucleotide gap; (iv) ␤-pol or another polymerase fills in the single-nucleotide gap; and (v) DNA ligase seals the gap (6). 5Ј-Terminal dRP might eventually undergo an uncatalyzed ␤-elimination reaction to generate a single-nucleotide gap (half-life Ϸ 2 h) (7), but this reaction is probably too slow to be relied upon in vivo. Coordination of steps (iii) and (iv) by ␤-pol presumably would lead to more efficient DNA repair. Thus, reaction (iii) assures an adequate substrate for ␤-pol, an enzyme without an intrinsic 5Ј-exonuclease activity.Proteolysis studies have revealed a two-domain structure for rat ␤-pol; independent digestions with four proteases resulted in the cleavage of the enzyme within a protease-sensitive region between residues 82 and 86 (8). Treatment with trypsin, for example, yielded an 8-kDa amino-terminal fragment (residues 3-75) and a 31-kDa carboxyl-terminal fragment (residues 87-334). In addition to its recently discovered dRpase activity, the 8-kDa domain (residues 1-87) is known to bind singlestranded DNA with an affinity similar to that of the intact enzyme (8). The 31-kDa domain (residues 88 -334) binds double-stranded DNA but not single-strand...

Down-regulation of DNA polymerase β accompanies somatic hypermutation in human BL2 cell lines

“…These enzymes were evaluated because it has been shown that pol ι is involved in V-region mutagenesis in human BL2 cells [3,16,32,33], and, in addition pol λ another member of the group of BER proficient DNA polymerases [35] can serve as a backup enzyme for pol β in BER [21]. Both of these polymerases carry intrinsic dRP lyase activities that may be important in short patch BER [24,[36][37][38]. We analyzed expression of these enzymes by immunoblotting and found no significant differences in the BL2 clones studied here.…”

Early steps in the DNA base excision/single-strand interruption repair pathway in mammalian cells

“…The choice of LP-BER vs. SN-BER may thus depend on, among other factors, the state of the 5′-dRP terminal moiety. With unaltered aldehyde group in deoxyribose Polb could carry out SN-BER by excising the 5′-dRP [17,81]. On the other hand, LP-BER is necessary when the AP sites are further oxidized by ROS, and the resulting 5′ blocking groups after cleavage of the DNA strand by APE could not be removed by Polb's 5′ end cleaning lyase activity.…”

Dynamics and diversions in base excision DNA repair of oxidized abasic lesions

“…In mammalian cells, most dRp excision is attributable to Polb (Sobol et al, 1996), specifically its amino-terminal 8-kilodalton domain (Matsumoto and Kim, 1995). In this process, lysine-72 performs a nucleophilic attack on the C1-carbonyl carbon of the 5'-terminal AP site, which forms a Schiff base intermediate between the enzyme and the DNA (Feng et al, 1998;Piersen et al, 1996) (Figure 2). belimination breaks the 3' phosphodiester, and the enzyme-deoxyribose complex is hydrolyzed to yield the free abasic-5-phosphate residue.…”