No abstract

During DNA synthesis, most DNA polymerases and reverse transcriptases select against ribonucleotides via a steric clash between the ribose 2′-hydroxyl group and the bulky side chain of an active site residue. Here, we demonstrated that human DNA polymerase λ used a novel sugar selection mechanism to discriminate against ribonucleotides, whereby the ribose 2′-hydroxyl group was excluded mostly by a backbone segment and slightly by the side chain of Y505. Such a steric clash was further demonstrated to be dependent on the size and orientation of the substituent covalently attached at the ribonucleotide C2′ position.

DNA repair pathways are essential for maintaining genome stability. DNA polymerase ␤ plays a critical role in base-excision repair in vivo. DNA polymerase , a recently identified X-family homolog of DNA polymerase ␤, is hypothesized to be a second polymerase involved in base-excision repair. The full-length DNA polymerase is comprised of three domains: a C-terminal DNA polymerase ␤-like domain, an N-terminal BRCA1 C-terminal domain, and a previously uncharacterized proline-rich domain. Strikingly, pre-steady-state kinetic analyses reveal that, although human DNA polymerase has almost identical fidelity to human DNA polymerase ␤, the C-terminal DNA polymerase ␤-like domain alone displays a dramatic, up to 100-fold loss in fidelity. We further demonstrate that the non-enzymatic proline-rich domain confers the increase in fidelity of DNA polymerase by significantly lowering incorporation rate constants of incorrect nucleotides. Our studies illustrate a novel mechanism, in which the DNA polymerase fidelity is controlled not by an accessory protein or a proofreading exonuclease domain but by an internal regulatory domain.A growing number of polymerases have been identified recently and many are hypothesized to function in DNA repair pathways. One of these polymerases is DNA polymerase (pol), 3 a new member of the X-family DNA polymerases (1-3). This family of DNA polymerases is a subdivision of a larger superfamily of nucleotidyltransferases (4). Human DNA polymerase is encoded by a gene located in human chromosome 10 (2, 3). The N terminus of the full-length pol (fpol) is composed of a nuclear localization signal motif, a breast cancer susceptibility protein BRCA1 C-terminal (BRCT) domain, and a proline-rich domain (see Fig. 1). BRCT domains are known to mediate protein-protein and protein-DNA interactions in DNA repair mechanisms and cell-cycle checkpoint regulation upon DNA damage (5). The proline-rich domain, which contains multiple serine, threonine, and proline residues, is found to functionally suppress the polymerase activity of pol (6) and limit strand-displacement synthesis (7). The C terminus of pol (tPol in Fig. 1) possesses both 5Ј-deoxyribose-5-phosphate lyase and DNA polymerase activities while sharing 33% sequence identity with DNA polymerase ␤ (pol␤) (1)(2)(3)8). pol␤, on the other hand, is also an X-family polymerase and is known to be involved in base excision repair (BER) pathways in vivo (9, 10). The x-ray crystal structures of tpol (11, 12) display a high degree of similarity with the corresponding subdomains of pol␤ (13,14), including the deoxyribose-5-phosphate lyase (also known as the 8-kDa domain), fingers, palm, and thumb subdomains. A comparison of the crystal structures of tpol⅐single-nucleotide gapped DNA, tpol⅐single-nucleotide gapped DNA⅐ddTTP, and tpol⅐nicked DNA⅐pyrophosphate suggests that no major protein domain movement occurs during catalysis (11). pol, like pol␤, lacks 3Ј35Ј exonuclease activity (1-3) and possesses low processivity when copying non-gapped DNA (8).At present, the ...

During short-patch base excision repair, the excision of a 5-terminal 2-deoxyribose-5-phosphate moiety of the downstream strand by the 5-2-deoxyribose-5-phosphate lyase activity of either DNA polymerase ␤ or is believed to occur after each respective enzyme catalyzes gap-filling DNA synthesis. Yet the effects of this 5-terminal 2-deoxyribose-5-phosphate moiety on the polymerase activities of these two enzymes have never been quantitatively determined. Moreover, x-ray crystal structures of truncated polymerase have revealed that the downstream strand and its 5-phosphate group of gapped DNA interact intensely with the dRPase domain, but the kinetic effect of these interactions is unclear. Here, we utilized pre-steady state kinetic methods to systematically investigate the effect of a downstream strand and its 5-moieties on the polymerase activity of the full-length human polymerase . The downstream strand and its 5-phosphate were both found to increase nucleotide incorporation efficiency (k p /K d ) by 15-and 11-fold, respectively, with the increase procured by the effect on the nucleotide incorporation rate constant k p rather than the ground state nucleotide binding affinity K d . With 4 single nucleotide-gapped DNA substrates containing a 1,2-dideoxyribose-5-phosphate moiety, a 2-deoxyribose-5-phosphate mimic, we measured the incorporation efficiencies of 16 possible nucleotides. Our results demonstrate that although this 5-terminal 2-deoxyribose-5-phosphate mimic does not affect the fidelity of polymerase , it moderately decreased the polymerase efficiency by 3.4-fold. Moreover, this decrease in polymerase efficiency is due to a drop of similar magnitude in k p rather than K d . The implication of the downstream strand and its 5-moieties on the kinetics of gapfilling synthesis is discussed.In mammalian cells, single base lesions are the most common form of DNA damage that arises either from exogenous DNAdamaging agents (1) or from endogenous biological processes resulting in base alkylation (2, 3), base oxidation (4), spontaneous cytosine deamination (3), and hydrolytic base loss (3,5,6). Base excision repair (BER) 3 is the major pathway to repair single base lesions (7). Short-patch and long-patch BER are the two subpathways of BER that remove and replace 1 (8 -10) and 2-11 nucleotides (11-13), respectively. Short-patch BER starts with the excision of a modified base by a DNA glycosylase, leaving a noncoding apurinic or apyrimidinic site in DNA. This lesion is further processed and repaired by a 5Ј-acting apurinic or apyrimidinic endonuclease, a DNA polymerase, a 5Ј-2-deoxyribose-5-phosphate lyase (dRPase), and a DNA ligase (12, 14 -16). It has been established that in mammalian systems DNA polymerase ␤ (Fig. 1, Pol␤), an X-family DNA polymerase, plays a critical role in short-patch BER (8, 9). The polymerase activity of Pol␤ catalyzes single nucleotide gap-filling synthesis (17), while its dRPase activity removes the 5Ј-terminal 2-deoxyribose-5-phosphate moiety (dRP) of a downstream strand (18). The uraci...