Template strand scrunching during DNA gap repair synthesis by human polymerase λ (original) (raw)
Related papers
Biochemistry, 1997
Mammalian DNA polymerase (pol ) is a small (39 kDa) DNA gap-filling enzyme that comprises an amino-terminal 8-kDa domain and a carboxy-terminal 31-kDa domain. In the work reported here, crystal structures of human pol complexed with blunt-ended segments of DNA show that, although the crystals belong to a different space group, the DNA is nevertheless bound in the pol binding channel in the same way as the DNA in previously reported structures of rat pol complexed with a templateprimer and ddCTP [Pelletier, H.The 8-kDa domain is in one of three previously observed positions relative to the 31-kDa domain, suggesting that the 8-kDa domain may assume only a small number of stable conformations. The thumb subdomain is in a more open position in the human pol -DNA binary complex than it is in the rat pol -DNA-ddCTP ternary complex, and a closing thumb upon nucleotide binding could represent the rate-limiting conformational change that has been observed in pre-steady-state kinetic studies. Intermolecular contacts between the DNA and the 8-kDa domain of a symmetry-related pol molecule reveal a plausible binding site on the 8-kDa domain for the downstream oligonucleotide of a gapped-DNA substrate; in addition to a lysine-rich binding pocket that accommodates a 5′-PO 4 end group, the 8-kDa domain also contains a newly discovered helix-hairpin-helix (HhH) motif that binds to DNA in the same way as does a structurally and sequentially homologous HhH motif in the 31-kDa domain. DNA binding by both HhH motifs is facilitated by a metal ion. In that HhH motifs have been identified in other DNA repair enzymes and DNA polymerases, the HhH-DNA interactions observed in pol may be applicable to a broad range of DNA binding proteins. The sequence similarity between the HhH motif of endonuclease III from Escherichia coli and the HhH motif of the 8-kDa domain of pol is particularly striking in that all of the conserved residues are clustered in one short sequence segment, LPGVGXK, where LPGV corresponds to a type II -turn (the hairpin turn), and GXK corresponds to a part of the HhH motif that is proposed to be critical for DNA binding and catalysis for both enzymes. These results suggest that endonuclease III and the 8-kDa domain of pol may employ a similar mode of DNA binding and may have similar catalytic mechanisms for their respective DNA lyase activities. A model for productive binding of pol to a gapped-DNA substrate requires a 90°bend in the singlestranded template, which could enhance nucleotide selectivity during DNA repair or replication. †
Time-lapse crystallography snapshots of a double-strand break repair polymerase in action
Nature Communications, 2017
DNA polymerase (pol) μ is a DNA-dependent polymerase that incorporates nucleotides during gap-filling synthesis in the non-homologous end-joining pathway of double-strand break repair. Here we report time-lapse X-ray crystallography snapshots of catalytic events during gap-filling DNA synthesis by pol μ. Unique catalytic intermediates and active site conformational changes that underlie catalysis are uncovered, and a transient third (product) metal ion is observed in the product state. The product manganese coordinates phosphate oxygens of the inserted nucleotide and PPi. The product metal is not observed during DNA synthesis in the presence of magnesium. Kinetic analyses indicate that manganese increases the rate constant for deoxynucleoside 5′-triphosphate insertion compared to magnesium. The likely product stabilization role of the manganese product metal in pol μ is discussed. These observations provide insight on structural attributes of this X-family double-strand break repair...
Structural Analysis of Strand Misalignment during DNA Synthesis by a Human DNA Polymerase
Cell, 2006
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 l, 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 l 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.
Structural insight into the substrate specificity of DNA Polymerase μ
Nature Structural & Molecular Biology, 2006
DNA polymerase l (Pol l) is a family X enzyme with unique substrate specificity that contributes to its specialized role in nonhomologous DNA end joining (NHEJ). To investigate Pol l's unusual substrate specificity, we describe the 2.4 Å crystal structure of the polymerase domain of murine Pol l bound to gapped DNA with a correct dNTP at the active site. This structure reveals substrate interactions with side chains in Pol l that differ from other family X members. For example, a single amino acid substitution, H329A, has little effect on template-dependent synthesis by Pol l 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.
Molecular and cellular function analysis of DNA polymerase X involved in DNA repair
2012
Single-nucleotide (1-nt) gaps in DNA can arise during various DNA processing events, particularly during base excision repair (BER). These lesions are repaired by the X-family DNA polymerases (PolXs) with high gap-filling activity. Some PolXs can bind productively to deoxyribonucleotides (dNTPs) in the absence of DNA and fill these 1-nt gaps. Although PolXs play a crucial role in efficient gap filling, currently, little is known about the kinetic and structural details of their productive dNTP binding. Here, I show that Thermus thermophilus HB8 PolX (ttPolX) has a strong binding affinity for Mg 2+-dNTPs in the absence of DNA, and that it follows a Theorell-Chance (hit-and-run) mechanism, with nucleotide binding as the first step. Comparison of the crystal structures of ttPolX in a binary complex, with dGTP, and in a ternary complex, with 1-nt gapped DNA and dideoxyguanosine 5'-triphosphate (ddGTP), revealed that the conformation of the incoming nucleotide depended on whether or not DNA was present. Furthermore, the Lys-263 residue located between two guanosine conformations was essential for the strong binding affinity of the enzyme. The ability to bind to either syn-dNTP or anti-dNTP and the involvement of a Theorell-Chance mechanism are key aspects of the strong nucleotide-binding and efficient gap-filling activities of PolXs. Furthermore, many bacterial PolXs have a polymerase and a histidinol phosphatase (PHP) domain at their C-termini, in addition to a PolX core (POLXc) domain, and possess 3'-5' exonuclease activity. I found that the PHP domain of ttPolX functions as three types of phosphoesterases, including a 3'-5' exonuclease, an apurinic/apyrimidinic (AP) endonuclease, and a 3'-phosphatase. The phosphoesterase and gap-filling activities described above are required for BER. Partial reconstitution of BER using T. thermophilus HB8 cell lysates revealed that the majority of the AP endonuclease and 3'-phosphatase activities are attributable to the phosphoesterase, endonuclease IV (EndoIV). However, ttPolX had sufficient 3'-phosphatase activity in EndoIV-deficient cells, indicating complementation. Furthermore, ttPolX was found to be the only efficient gap-filling DNA polymerase in the cell. These results indicate that ttPolX is a multifunctional enzyme that is specialized in BER.
Biochemical and Biophysical Research Communications, 2004
Non-homologous end joining (NHEJ) is one of two pathways responsible for the repair of double-strand breaks in eukaryotic cells. The mechanism involves the alignment of broken DNA ends with minimal homology, fill in of short gaps by DNA polymerase(s), and ligation by XRCC4-DNA ligase IV complex. The gap-filling polymerase has not yet been positively identified, but recent biochemical studies have implicated DNA polymerase k (pol k), a novel DNA polymerase that has been assigned to the pol X family, in this process. Here we demonstrate that purified pol k can efficiently catalyze gap-filling synthesis on DNA substrates mimicking NHEJ. By designing two truncated forms of pol k, we also show that the unique proline-rich region in pol k plays a role in limiting strand displacement synthesis, a feature that may help its participation in in vivo NHEJ. Moreover, pol k interacts with XRCC4-DNA ligase IV via its N-terminal BRCT domain and the interaction stimulates the DNA synthesis activity of pol k. Taken together, these data strongly support that pol k functions in DNA polymerization events during NHEJ.
Molecular basis for DNA repair synthesis on short gaps by mycobacterial Primase-Polymerase C
Nature Communications
Cells utilise specialized polymerases from the Primase-Polymerase (Prim-Pol) superfamily to maintain genome stability. Prim-Pol's function in genome maintenance pathways including replication, repair and damage tolerance. Mycobacteria contain multiple Prim-Pols required for lesion repair, including Prim-PolC that performs short gap repair synthesis during excision repair. To understand the molecular basis of Prim-PolC's gap recognition and synthesis activities, we elucidated crystal structures of pre-and post-catalytic complexes bound to gapped DNA substrates. These intermediates explain its binding preference for short gaps and reveal a distinctive modus operandi called Synthesis-dependent Template Displacement (STD). This mechanism enables Prim-PolC to couple primer extension with template base dislocation, ensuring that the unpaired templating bases in the gap are ushered into the active site in an ordered manner. Insights provided by these structures establishes the molecular basis of Prim-PolC's gap recognition and extension activities, while also illuminating the mechanisms of primer extension utilised by closely related Prim-Pols.