Structure-function studies of DNA polymerase λ.
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ABSTRACT:
SUBMITTER: Bebenek K
PROVIDER: S-EPMC4018081 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:DNA polymerase lambda (Pol lambda) is one of several DNA polymerases suggested to participate in base excision repair (BER), in repair of broken DNA ends and in translesion synthesis. It has been proposed that the nature of the DNA intermediates partly determines which polymerase is used for a particular repair reaction. To test this hypothesis, here we examine the ability of human Pol lambda to extend mismatched primer-termini, either on 'open' template-primer substrates, or on its preferred substrate, a 1 nt gapped-DNA molecule having a 5'-phosphate. Interestingly, Pol lambda extended mismatches with an average efficiency of approximately 10(-2) relative to matched base pairs. The match and mismatch extension catalytic efficiencies obtained on gapped molecules were approximately 260-fold higher than on template-primer molecules. A crystal structure of Pol lambda in complex with a single-nucleotide gap containing a dG.dGMP mismatch at the primer-terminus (2.40 A) suggests that, at least for certain mispairs, Pol lambda is unable to differentiate between matched and mismatched termini during the DNA binding step, thus accounting for the relatively high efficiency of mismatch extension. This property of Pol lambda suggests a potential role as a 'mismatch extender' during non-homologous end joining (NHEJ), and possibly during translesion synthesis.
| S-EPMC1904104 | biostudies-literature
Project description:Base excision repair (BER) plays a vital role in maintaining genomic integrity in mammalian cells. DNA polymerase λ (Pol λ) is believed to play a backup role to DNA polymerase β (Pol β) in base excision repair. Two oxidized abasic lesions that are produced by a variety of DNA-damaging agents, including several antitumor antibiotics, the C4'-oxidized abasic site following Ape1 incision (pC4-AP), and 5'-(2-phosphoryl-1,4-dioxobutane) (DOB), irreversibly inactivate Pol β and Pol λ. The interactions of DOB and pC4-AP with Pol λ are examined in detail using DNA substrates containing these lesions at defined sites. Single-turnover kinetic experiments show that Pol λ excises DOB almost 13 times more slowly than a 5'-phosphorylated 2-deoxyribose (dRP). pC4-AP is excised approximately twice as fast as DOB. The absolute rate constants are considerably slower than those reported for Pol β for the respective reactions, suggesting that Pol λ may be an inefficient backup in BER. DOB inactivates Pol λ approximately 3-fold less efficiently than it does Pol β, and the difference can be attributed to a higher K(I) (33 ± 7 nM). Inactivation of Pol λ's lyase activity by DOB also prevents the enzyme from conducting polymerization following preincubation of the protein and DNA. Mass spectral analysis of GluC-digested Pol λ inactivated by DOB shows that Lys324 is modified. There is inferential support for the idea that Lys312 may also be modified. Both residues are within the Pol λ lyase active site. When acting on pC4-AP, Pol λ achieves approximately four turnovers on average before being inactivated. Lyase inactivation by pC4-AP is also accompanied by loss of polymerase activity, and mass spectrometry indicates that Lys312 and Lys324 are modified by the lesion. The ability of DOB and pC4-AP to inactivate Pol λ provides additional evidence that these lesions are significant sources of the cytotoxicity of DNA-damaging agents that produce them.
| S-EPMC3566640 | biostudies-literature
Project description:The anticancer activity of cytarabine (AraC) and gemcitabine (dFdC) is thought to result from chain termination after incorporation into DNA. To investigate their incorporation into DNA at atomic level resolution, we present crystal structures of human DNA polymerase lambda (Pol lambda) bound to gapped DNA and containing either AraC or dFdC paired opposite template dG. These structures reveal that AraC and dFdC can bind within the nascent base pair binding pocket of Pol lambda. Although the conformation of the ribose of AraCTP is similar to that of normal dCTP, the conformation of dFdCTP is significantly different. Consistent with these structures, Pol lambda efficiently incorporates AraCTP but not dFdCTP. The data are consistent with the possibility that Pol lambda could modulate the cytotoxic effect of AraC.
| S-EPMC2878035 | biostudies-literature
Project description:Differences in the substrate specificity of mammalian family X DNA polymerases are proposed to partly depend on a loop (loop 1) upstream of the polymerase active site. To examine if this is the case in DNA polymerase λ (pol λ), here we characterize a variant of the human polymerase in which nine residues of loop 1 are replaced with four residues from the equivalent position in pol β. Crystal structures of the mutant enzyme bound to gapped DNA with and without a correct dNTP reveal that the change in loop 1 does not affect the overall structure of the protein. Consistent with these structural data, the mutant enzyme has relatively normal catalytic efficiency for correct incorporation, and it efficiently participates in non-homologous end joining of double-strand DNA breaks. However, DNA junctions recovered from end-joining reactions are more diverse than normal, and the mutant enzyme is substantially less accurate than wild-type pol λ in three different biochemical assays. Comparisons of the binary and ternary complex crystal structures of mutant and wild-type pol λ suggest that loop 1 modulates pol λ's fidelity by controlling dNTP-induced movements of the template strand and the primer-terminal 3'-OH as the enzyme transitions from an inactive to an active conformation.
| S-EPMC2938210 | biostudies-literature
Project description:The X family of DNA polymerases in eukaryotic cells consists of terminal transferase and DNA polymerases beta, lambda, and mu. These enzymes have similar structural portraits, yet different biochemical properties, especially in their interactions with DNA. None of these enzymes possesses a proofreading subdomain, and their intrinsic fidelity of DNA synthesis is much lower than that of a polymerase that functions in cellular DNA replication. In this review, we discuss the similarities and differences of three members of Family X: polymerases beta, lambda, and mu. We focus on biochemical mechanisms, structural variation, fidelity and lesion bypass mechanisms, and cellular roles. Remarkably, although these enzymes have similar three-dimensional structures, their biochemical properties and cellular functions differ in important ways that impact cellular function.
| S-EPMC2846199 | biostudies-literature
Project description:By extending synthesis opposite from a diverse array of DNA lesions, DNA polymerase (Pol) ζ performs a crucial role in translesion synthesis (TLS). In yeast and cancer cells, Rev1 functions as an indispensable scaffolding component of Polζ and it imposes highly error-prone TLS upon Polζ. However, for TLS that occurs during replication in normal human cells, Rev1 functions instead as a scaffolding component of Pols η, ι, and κ and Rev1-dependent TLS by these Pols operates in a predominantly error-free manner. The lack of Rev1 requirement for Polζ function in TLS in normal cells suggested that some other protein substitutes for this Rev1 role. Here, we identify a novel role of Polλ as an indispensable scaffolding component of Polζ. TLS studies opposite a number of DNA lesions support the conclusion that as an integral component, Polλ adapts Polζ-dependent TLS to operate in a predominantly error-free manner in human cells, essential for genome integrity and cellular homeostasis.
| S-EPMC7898466 | biostudies-literature
Project description:The simple deletion of nucleotides is common in many organisms. It can be advantageous when it activates genes beneficial to microbial survival in adverse environments, and deleterious when it mutates genes relevant to survival, cancer or degenerative diseases. The classical idea is that simple deletions arise by strand slippage. A prime opportunity for slippage occurs during DNA synthesis, but it remains unclear how slippage is controlled during a polymerization cycle. Here, we report crystal structures and molecular dynamics simulations of mutant derivatives of DNA polymerase lambda bound to a primer-template during strand slippage. Relative to the primer strand, the template strand is in multiple conformations, indicating intermediates on the pathway to deletion mutagenesis. Consistent with these intermediates, the mutant polymerases generate single-base deletions at high rates. The results indicate that dNTP-induced template strand repositioning during conformational rearrangements in the catalytic cycle is crucial to controlling the rate of strand slippage.
| S-EPMC2278112 | biostudies-literature
Project description:It has now been nearly two decades since the first solution structures of DNA duplexes covalently damaged by metabolically activated polycyclic aromatic hydrocarbons and amines were determined by NMR. Dozens of such high-resolution structures are now available, and some broad structural themes have been uncovered. It has been hypothesized that the solution structures are relevant to the biochemical processing of the adducts. The structural features of the adducts are considered to determine their mutational properties in DNA polymerases and their repair susceptibilities. In recent years, a number of crystal structures of DNA adducts of interest to our work have been determined in DNA polymerases. Accordingly, it is now timely to consider how NMR solution structures relate to structures within DNA polymerases. The NMR solution structural themes for polycyclic aromatic adducts are often observed in polymerase crystal structures. While the polymerase interactions can on occasion override the solution preferences, intrinsic adduct conformations favored in solution are often manifested within polymerases and likely play a significant role in lesion processing.
| S-EPMC4692375 | biostudies-literature
Project description:DNA polymerase (dpol) β has served as a model for structural, kinetic, and computational characterization of the DNA synthesis reaction. The laboratory directed by Samuel H. Wilson has utilized a multifunctional approach to analyze the function of this enzyme at the biological, chemical, and molecular levels for nearly 50 years. Over this time, it has become evident that correlating static crystallographic structures of dpol β with solution kinetic measurements is a daunting task. However, aided by computational and spectroscopic approaches, novel and unexpected insights have emerged. While dpols generally insert wrong nucleotides with similar poor efficiencies, their capacity to insert the right nucleotide depends on the identity of the dpol. Accordingly, the ability to choose right from wrong depends on the efficiency of right, rather than wrong, nucleotide insertion. Structures of dpol β in various liganded forms published by the Wilson laboratory, and others, have provided molecular insights into the molecular attributes that hasten correct nucleotide insertion and deter incorrect nucleotide insertion. Computational approaches have bridged the gap between structures of intermediate complexes and provided insights into this basic and essential chemical reaction.
| S-EPMC7643811 | biostudies-literature
Project description:DNA polymerases play a crucial role in the cell cycle due to their involvement in genome replication and repair. Understanding the reaction mechanism by which these polymerases carry out their function can provide insights into these processes. Recently, the crystal structures of human DNA polymerase lambda (Pollambda) have been reported both for pre- and post-catalytic complexes [García-Díaz et al., DNA Repair 3 (2007), 1333]. Here we employ the pre-catalytic complex as a starting structure for the determination of the catalytic mechanism of Pollambda using ab initio quantum mechanical/molecular mechanical methods. The reaction path has been calculated using Mg(2+) and Mn(2+) as the catalytic metals. In both cases the reaction proceeds through a two-step mechanism where the 3'-OH of the primer sugar ring is deprotonated by one of the conserved Asp residues (D490) in the active site before the incorporation of the nucleotide to the nascent DNA chain. A significant charge transfer is observed between both metals and some residues in the active site as the reaction proceeds. The optimized reactant and product structures agree with the reported crystal structures. In addition, the calculated reaction barriers for both metals are close to experimentally estimated barriers. Energy decomposition analysis to explain individual residue contributions suggests that several amino acids surrounding the active site are important for catalysis. Some of these residues, including R420, R488 and E529, have been implicated in catalysis by previous mutagenesis experiments on the homologous residues on Polbeta. Furthermore, Pollambda residues R420 and E529 found to be important from the energy decomposition analysis, are homologous to residues R183 and E295 in Polbeta, both of which are linked to cancer. In addition, residues R386, E391, K422 and K472 appear to have an important role in catalysis and could be a potential target for mutagenesis experiments. There is partial conservation of these residues across the Pol X family of DNA polymerases.
| S-EPMC2586819 | biostudies-literature