Functional Analysis of the Amino-terminal 8-kDa Domain of DNA Polymerase β as Revealed by Site-directed Mutagenesis

Mitochondrial DNA polymerase ␥ (pol ␥) is active in base excision repair of AP (apurinic/apyrimidinic) sites in DNA. Usually AP site repair involves cleavage on the 5 side of the deoxyribose phosphate by AP endonuclease. Previous experiments suggested that DNA pol ␥ acts to catalyze the removal of a 5-deoxyribose phosphate (dRP) group in addition to playing the conventional role of a DNA polymerase. We confirm that DNA pol ␥ is an active dRP lyase and show that other members of the family A of DNA polymerases including Escherichia coli DNA pol I also possess this activity. The dRP lyase reaction proceeds by formation of a covalent enzyme-DNA intermediate that is converted to an enzyme-dRP intermediate following elimination of the DNA. Both intermediates can be cross-linked with NaBH 4 . For both DNA pol ␥ and the Klenow fragment of pol I, the enzyme-dRP intermediate is extremely stable. This limits the overall catalytic rate of the dRP lyase, so that family A DNA polymerases, unlike pol ␤, may only be able to act as dRP lyases in repair of AP sites when they occur at low frequency in DNA. Abasic (AP)1 sites in DNA are produced frequently by spontaneous base loss or by the action of DNA glycosylases that initiate base excision repair of damaged bases in DNA (1, 2). If AP sites are not repaired quickly, DNA polymerases are capable of replicating through these non-instructional lesions, resulting in frequent misincorporation. Cells have adapted vigorous mechanisms for the repair of AP sites, usually beginning with incision of the phosphodiester backbone on the 5Ј side of the lesion by AP endonuclease to produce a 3Ј-OH terminus adjacent to a 5Ј-deoxyribose phosphate, or 5Ј-dRP group (3, 4). The 3Ј-OH terminus provides a primer for repair synthesis by DNA polymerase. The 5Ј-dRP moiety must be removed in the course of repair. This is frequently accomplished by a ␤-elimination reaction catalyzed by an AP lyase activity. When an AP lyase acts on an exposed 5Ј-dRP residue produced by a class II AP endonuclease, the activity may be considered as a dRP lyase. The dRP lyase mechanism involves nucleophilic attack on the C-1 position of the 5Ј-dRP group by a free amino group of the enzyme (5). This produces a transient covalent intermediate in which the DNA substrate is linked to the enzyme as a Schiff base that can be stabilized by treatment with strong reducing agents, such as sodium borohydride. This borohydride trapping reaction has been used to identify a number of DNA repair enzymes with AP lyase activity, including several DNA glycosylases (6, 7).Eukaryotic cells have redundant pathways for repairing AP sites in nuclear DNA. Under most circumstances, AP sites are repaired by a pathway that employs the dedicated repair polymerase, DNA pol ␤. Recently, Matsumoto and Kim (8) showed that the pol ␤ is especially well adapted to function in base excision repair, since it contains a dRP lyase activity in a small domain not required for polymerase activity. The active site of the pol ␤ dRP lyase has been localized to a heli...

Section: Abasic (Ap)mentioning

confidence: 92%

Characterization of a Catalytically Slow AP Lyase Activity in DNA Polymerase γ and Other Family A DNA Polymerases

Pinz

Bogenhagen

2000

“…The 5Ј-phosphate binds near a lysine-rich pocket (blue) and the proposed lyase catalytic center of the 8-kDa domain. Structural and sitedirected mutagenesis data suggest that Lys 35 and Lys 68 interact with this phosphate, and Lys 72 has been proposed to be in the dRP lyase active site of the 8-kDa domain (34,35). Nearby lysine residues Lys 60 and Lys 68 are also shown.…”

Section: Methodsmentioning

confidence: 99%

“…These investigators showed that a Schiff base intermediate is formed between the dRP-containing DNA substrate and the enzyme. The Schiff base nucleophile in the 8-kDa domain has been suggested to be Lys 72 by site-directed mutagenesis (34,35). This residue is in close proximity to the 5Ј-phosphate product group in the gapped DNA/enzyme crystal structure (26) and is part of the putative dRP lyase active site identified in NMR structures (23) of the 8-kDa domain (Fig.…”

mentioning

confidence: 99%

Mapping of the 5′-2-Deoxyribose-5-phosphate Lyase Active Site in DNA Polymerase β by Mass Spectrometry

Deterding

Prasad

Mullen

et al. 2000

Self Cite

The mechanism of the 5-2-deoxyribose-5-phosphate lyase reaction catalyzed by mammalian DNA ␤-polymerase (␤-pol) was investigated using a cross-linking methodology in combination with mass spectrometric analyses. The approach included proteolysis of the covalently cross-linked protein-DNA complex with trypsin, followed by isolation, peptide mapping, and mass spectrometric and tandem mass spectrometric analyses. The 8-kDa domain of ␤-pol was covalently cross-linked to a 5-2-deoxyribose-5-phosphate-containing DNA substrate by sodium borohydride reduction. Using tandem mass spectrometry, the location of the DNA adduct on the 8-kDa domain was unequivocally determined to be at the Lys 72 residue. No additional amino acid residues were found as minor cross-linked species. These data allow assignment of Lys 72 as the sole Schiff base nucleophile in the 8-kDa domain of ␤-pol. These results provide the first direct evidence in support of a catalytic mechanism involving nucleophilic attack by Lys 72 at the abasic site.Mammalian DNA polymerase ␤ (␤-pol), 1 a constitutively expressed "housekeeping" enzyme, has been implicated in DNA base excision repair (BER) (1-4). Base excision repair is thought to be the major repair pathway protecting cells against single base DNA damage. Recent evidence has indicated that BER in mammalian cells is mediated through at least two subpathways that are differentiated by the patch sizes and by the enzymes involved (5-8). These subpathways are designated as "single nucleotide" BER and long patch BER (two to several nucleotide repair patches). The single nucleotide BER subpathway is a sequential multistep process, where ␤-pol is involved in two steps (9, 10). The overall scheme for the single nucleotide BER subpathway can be outlined as follows: (i) recognition and cleavage of an altered/damaged base by a specific monofunctional DNA glycosylase (a glycosylase lacking intrinsic apurinic/apyrimidinic (AP) lyase activity) resulting in an AP site; (ii) class II AP endonuclease incision of the phosphodiester backbone 5Ј to the AP site, resulting in 3Ј-hydroxyl and 5Ј-2-deoxyribose-5-phosphate (dRP) containing termini; (iii) replacement of the missing base; (iv) removal of the 5Ј-sugar phosphate to provide a 5Ј-phosphate for DNA ligation by ␤-pol; and (v) DNA ligase sealing of the nick (3, 9 -13).␤-Pol is a multifunctional enzyme consisting of an 8-kDa N-terminal domain with dRP lyase activity and a 31-kDa Cterminal domain with nucleotidyltransferase activity (9, 14 -16). These two domains appear to be packed together in ␤-pol in solution, as revealed by comparison of axial ratios of ␤-pol (5.0) and the isolated 8-and 31-kDa domains (2.3 and 5.5, respectively) (17). Circular dichroism analysis has revealed that the 8-kDa domain is essentially ␣-helical in nature (16), and NMR solution and crystal structures (18, 19) have since confirmed the predominance of this secondary structure.The 8-kDa domain of ␤-pol (residues 1-87), originally characterized as a single-stranded DNA binding domain, is formed fr...

“…For example, TdT is a template-independent polymerase involved in antigen receptor diversification during V(D)J recombination (11,12). In contrast Pol ␤ is a moderately faithful, template-dependent enzyme (13,14) that, in addition to its polymerase activity, possesses a 5Ј, 2Ј-deoxyribose-5-phosphate (dRP) lyase activity (15,16). Finally Pol , likely involved in non-homologous endjoining (NHEJ) (17), is a largely template-dependent polymerase that is also endowed with some template-independent polymerization activity (9).…”

mentioning

confidence: 99%

The Frameshift Infidelity of Human DNA Polymerase λ

Bębenek

García‐Díaz

Blanco

et al. 2003

110

145

DNA polymerase(Pol ) is a member of the Pol X family having properties in common with several other mammalian DNA polymerases. To obtain clues to possible functions in vivo, we have determined the fidelity of DNA synthesis by human Pol . The results indicate that the average single-base deletion error rate of Pol is higher than those of other mammalian polymerases. In fact, unlike other DNA polymerases, Pol generates single-base deletions at average rates that substantially exceed base substitution rates. Moreover, the sequence specificity for single-base deletions made by Pol is different from that of other DNA polymerases and reveals that Pol readily uses template-primers with limited base pair homology at the primer terminus. This ability, together with an ability to fill short gaps in DNA at low dNTP concentrations, is consistent with a role for mammalian Pol in non-homologous end-joining. This may include non-homologous end-joining of strand breaks resulting from DNA damage, because Pol has intrinsic 5 ,2 -deoxyribose-5-phosphate lyase activity.