Biomedical subjects

Miguel Garcia-Diaz

Publications and source records attributed to Miguel Garcia-Diaz.

12 recordsLinked to original sources

PubMed2006

Structural insight into the substrate specificity of DNA Polymerase mu.

DNA polymerase mu (Pol mu) is a family X enzyme with unique substrate specificity that contributes to its specialized role in nonhomologous DNA end joining (NHEJ). To investigate Pol mu's unusual substrate specificity, we describe the 2.4 A crystal structure of the polymerase domain of murine Pol mu bound to gapped DNA with a correct dNTP at the active site. This structure reveals substrate interactions with side chains in Pol mu that differ from other family X members. For example, a single amino acid substitution, H329A, has little effect on template-dependent synthesis by Pol mu from a paired primer terminus, but it reduces both template-independent and template-dependent synthesis during NHEJ of intermediates whose 3' ends lack complementary template strand nucleotides. These results provide insight into the substrate specificity and differing functions of four closely related mammalian family X DNA polymerases.

Amino Acid Sequence↗

PubMed2006

A unique error signature for human DNA polymerase nu.

Human DNA polymerase nu (pol nu) is one of three A family polymerases conserved in vertebrates. Although its biological functions are unknown, pol nu has been implicated in DNA repair and in translesion DNA synthesis (TLS). Pol nu lacks intrinsic exonucleolytic proofreading activity and discriminates poorly against misinsertion of dNTP opposite template thymine or guanine, implying that it should copy DNA with low base substitution fidelity. To test this prediction and to comprehensively examine pol nu DNA synthesis fidelity as a clue to its function, here we describe human pol nu error rates for all 12 single base-base mismatches and for insertion and deletion errors during synthesis to copy the lacZ alpha-complementation sequence in M13mp2 DNA. Pol nu copies this DNA with average single-base insertion and deletion error rates of 7 x 10(-5) and 17 x 10(-5), respectively. This accuracy is comparable to that of replicative polymerases in the B family, lower than that of its A family homolog, human pol gamma, and much higher than that of Y family TLS polymerases. In contrast, the average single-base substitution error rate of human pol nu is 3.5 x 10(-3), which is inaccurate compared to the replicative polymerases and comparable to Y family polymerases. Interestingly, the vast majority of errors made by pol nu reflect stable misincorporation of dTMP opposite template G, at average rates that are much higher than for homologous A family members. This pol nu error is especially prevalent in sequence contexts wherein the template G is preceded by a C-G or G-C base pair, where error rates can exceed 10%. Amino acid sequence alignments based on the structures of more accurate A family polymerases suggest substantial differences in the O-helix of pol nu that could contribute to this unique error signature.

Amino Acid Sequence↗

PubMed2006

Mechanism of a genetic glissando: structural biology of indel mutations.

Insertions and deletions of bases in DNA (collectively termed "indels") are both common and biologically relevant, being associated with different human pathologies including cancer and diseases associated with expansions of repeats. Four decades of research have resulted in several hypotheses regarding how indels are generated during DNA synthesis and how they subsequently undergo or escape correction. Recent structural studies of DNA polymerases bound to mutagenic substrates have increased our understanding of how DNA polymerases cope with abnormal substrates. These structures provide insight into the molecular mechanisms underlying indel generation.

DNA↗

PubMed2006

Structural analysis of strand misalignment during DNA synthesis by a human DNA polymerase.

Insertions and deletions in coding sequences can alter the reading frame of genes and have profound biological consequences. In 1966, Streisinger proposed that these mutations result from strand slippage, which in repetitive sequences generates misaligned intermediates stabilized by correct base pairing that support polymerization. We report here crystal structures of human DNA polymerase lambda, which frequently generates deletion mutations, bound to such intermediates. Each contains an extrahelical template nucleotide upstream of the active site. Surprisingly, the extra nucleotide, even when combined with an adjacent mismatch, does not perturb polymerase active site geometry, which is indistinguishable from that for correctly aligned strands. These structures reveal how pol lambda can polymerize on substrates with minimal homology during repair of double-strand breaks and represent strand-slippage intermediates consistent with Streisinger's classical hypothesis. They are thus relevant to the origin of single-base deletions, a class of mutations that can confer strong biological phenotypes.

Binding Sites↗

PubMed2005

Structure-function studies of DNA polymerase lambda.

DNA polymerase lambda is a member of the X family of polymerases that is implicated in non-homologous end-joining of double-strand breaks in DNA and in base excision repair of DNA damage. To better understand the roles of DNA polymerase lambda in these repair pathways, here we review its structure and biochemical properties, with emphasis on its gap-filling polymerization activity, its dRP lyase activity and its unusual DNA synthetic (in)fidelity.

DNA Polymerase beta↗

PubMed2005

A gradient of template dependence defines distinct biological roles for family X polymerases in nonhomologous end joining.

Three Pol X family members have been linked to nonhomologous end joining (NHEJ) in mammals. Template-independent TdT promotes diversity during NHEJ-dependent repair of V(D)J recombination intermediates, but the roles of the template-dependent polymerases mu and lambda in NHEJ remain unclear. We show here that pol mu and pol lambda are similarly recruited by NHEJ factors to fill gaps when ends have partially complementary overhangs, suggesting equivalent roles promoting accuracy in NHEJ. However, only pol mu promotes accuracy during immunoglobulin kappa recombination. This distinctive in vivo role correlates with the TdT-like ability of pol mu, but not pol lambda, to act when primer termini lack complementary bases in the template strand. However, unlike TdT, synthesis by pol mu in this context is primarily instructed by a template from another DNA molecule. This apparent gradient of template dependence is largely attributable to a small structural element that is present but different in all three polymerases.

Amino Acid Sequence↗

PubMed2005

Biochemical properties of Saccharomyces cerevisiae DNA polymerase IV.

Although mammals encode multiple family X DNA polymerases implicated in DNA repair, Saccharomyces cerevisiae has only one, DNA polymerase IV (pol IV). To better understand the repair functions of pol IV, here we characterize its biochemical properties. Like mammalian pol beta and pol lambda, but not pol mu, pol IV has intrinsic 5'-2-deoxyribose-5-phosphate lyase activity. Pol IV has low processivity and can fill short gaps in DNA. Unlike the case with pol beta and pol lambda, the gap-filling activity of pol IV is not enhanced by a 5'-phosphate on the downstream primer but is stimulated by a 5'-terminal synthetic abasic site. Pol IV incorporates rNTPs into DNA with an unusually high efficiency relative to dNTPs, a property in common with pol mu but not pol beta or pol lambda. Finally, pol IV is highly inaccurate, with an unusual error specificity indicating the ability to extend primer termini with limited homology. These properties are consistent with a possible role for pol IV in base excision repair and with its known role in non-homologous end joining of double strand breaks, perhaps including those with damaged ends.

Amino Acid Sequence↗

PubMed2004

A closed conformation for the Pol lambda catalytic cycle.

Pol lambda is a family X member believed to fill short gaps during DNA repair. Here we report crystal structures of Pol lambda representing three steps in filling a single-nucleotide gap. These structures indicate that, unlike other DNA polymerases, Pol lambda does not undergo large subdomain movements during catalysis, and they provide a clear characterization of the geometry and stereochemistry of the in-line nucleotidyl transfer reaction.

Catalysis↗

PubMed2004

A structural solution for the DNA polymerase lambda-dependent repair of DNA gaps with minimal homology.

Human DNA polymerase lambda (Pol lambda) is a family X member with low frameshift fidelity that has been suggested to perform gap-filling DNA synthesis during base excision repair and during repair of broken ends with limited homology. Here, we present a 2.1 A crystal structure of the catalytic core of Pol lambda in complex with DNA containing a two nucleotide gap. Pol lambda makes limited contacts with the template strand at the polymerase active site, and superimposition with Pol beta in a ternary complex suggests a shift in the position of the DNA at the active site that is reminiscent of a deletion intermediate. Surprisingly, Pol lambda can adopt a closed conformation, even in the absence of dNTP binding. These observations have implications for the catalytic mechanism and putative DNA repair functions of Pol lambda.

Amino Acid Sequence↗

PubMed2003

Implication of DNA polymerase lambda in alignment-based gap filling for nonhomologous DNA end joining in human nuclear extracts.

Accurate repair of free radical-mediated DNA double-strand breaks by the nonhomologous end joining pathway requires replacement of fragmented nucleotides in the aligned ends by a gap-filling DNA polymerase. Nuclear extracts of human HeLa cells, supplemented with recombinant XRCC4-DNA ligase IV complex (XRCC4/ligase IV), were capable of accurately rejoining model double-strand break substrates with a 1- or 2-base gap, and the gap-filling step was dependent on XRCC4/ligase IV. To determine what polymerase was responsible for gap filling, end joining was examined in the presence of polyclonal antibodies against each of two prime candidate enzymes, DNA polymerases mu and lambda, both of which were present in the extracts. For a DNA substrate with partially complementary 3' overhangs and a 2-base gap, antibodies to polymerase lambda completely eliminated both gap filling and accurate end joining, whereas antibodies to polymerase mu had little effect. Immunodepletion of polymerase lambda, but not polymerase mu, likewise blocked both gap filling and end joining, and both functions could be restored by addition of recombinant polymerase lambda. Recombinant polymerase mu, and a truncated polymerase lambda lacking the Brca1 C-terminal domain, were at least 10-fold less active in restoring gap filling to the immunodepleted extracts, and polymerase beta was completely inactive. The results suggest that polymerase lambda is the primary gap-filling polymerase for accurate nonhomologous end joining, and that the Brca1 C-terminal domain is required for this activity.

Base Sequence↗

PubMed2003

Solution structure of the lyase domain of human DNA polymerase lambda.

DNA polymerase lambda (pol lambda) is a recently discovered nuclear enzyme belonging to the pol X family of DNA polymerases that exhibits a 32% sequence identity with the nuclear DNA repair protein, pol beta. Structural modeling suggests that pol lambda contains the palm, fingers, thumb, and 8 kDa lyase domains present in pol beta, as well as an additional N-terminal BRCT domain and a serine-proline-rich linker that are presumably involved in protein-protein interactions. The 8 kDa domain of pol beta is important for DNA binding and contains the dRP lyase activity, which is the rate-limiting step in the single-nucleotide base excision repair (BER) pathway of damaged DNA. Recently, it was shown that the 8 kDa domain of pol lambda also contains the dRP lyase activity. To gain further insight into the catalytic mechanism of dRP removal by pol lambda, we have determined the solution structure of the 8 kDa lyase domain of human DNA pol lambda via multidimensional NMR methods and the ARIA program. The resulting structures exhibited a high degree of similarity with the 8 kDa lyase domain of pol beta. Specifically, the side chains of residues W274, R275, Y279, K307, R308, and K312 are in similar positions to the functionally important side chains of residues H34, K35, Y39, K60, K68, and K72 in the 8 kDa lyase domain of pol beta. This suggests that, on the basis of the proposed roles of these residues in pol beta, the corresponding pol lambda side chains may be involved in DNA binding and dRP lyase activity. The structural alignment of W274 (pol lambda) with H34 (pol beta) indicates that the former is probably involved in a similar base stacking interaction with template DNA at the position of the gap, in contrast with several previous proposals which aligned D272 with H34. In a few cases for which there is a nonconservative substitution in the sequence alignment, a structural comparison shows a positionally and, hence, probably a functionally equivalent residue, e.g., K60 in pol beta and K307 in pol lambda. Additionally, on the basis of the structural alignment obtained, several previously proposed mechanistic hypotheses can be evaluated.

Amino Acid Sequence↗

PubMed2003

The frameshift infidelity of human DNA polymerase lambda. Implications for function.

DNA polymerase lambda (Pol lambda) 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 lambda. The results indicate that the average single-base deletion error rate of Pol lambda is higher than those of other mammalian polymerases. In fact, unlike other DNA polymerases, Pol lambda generates single-base deletions at average rates that substantially exceed base substitution rates. Moreover, the sequence specificity for single-base deletions made by Pol lambda is different from that of other DNA polymerases and reveals that Pol lambda 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 lambda in non-homologous end-joining. This may include non-homologous end-joining of strand breaks resulting from DNA damage, because Pol lambda has intrinsic 5',2'-deoxyribose-5-phosphate lyase activity.

Base Sequence↗