DNA and Chromosomes
DNA polymerase β nucleotide-stabilized template misalignment fidelity depends on local sequence contextDNA polymerase β has two DNA-binding domains that interact with the opposite sides of short DNA gaps. These domains contribute two activities that modify the 5′ and 3′ margins of gapped DNA during base excision repair. DNA gaps greater than 1 nucleotide (nt) pose an architectural and logistical problem for the two domains to interact with their respective DNA termini. Here, crystallographic and kinetic analyses of 2-nt gap-filling DNA synthesis revealed that the fidelity of DNA synthesis depends on local sequence context.
Long-term, genome-wide kinetic analysis of the effect of the circadian clock and transcription on the repair of cisplatin-DNA adducts in the mouse liverCisplatin is the most commonly used chemotherapeutic drug for managing solid tumors. However, toxicity and the innate or acquired resistance of cancer cells to the drug limit its usefulness. Cisplatin kills cells by forming cisplatin-DNA adducts, most commonly the Pt-d(GpG) diadduct. We recently showed that, in mice, repair of this adduct 2 h following injection is controlled by two circadian programs. 1) The circadian clock controls transcription of 2000 genes in liver and, via transcription-directed repair, controls repair of the transcribed strand (TS) of these genes in a rhythmic fashion unique to each gene’s phase of transcription.
The nature of the DNA substrate influences pre-catalytic conformational changes of DNA polymerase βDNA polymerase β (Pol β) is essential for maintaining genomic integrity. During short-patch base excision repair (BER), Pol β incorporates a nucleotide into a single-gapped DNA substrate. Pol β may also function in long-patch BER, where the DNA substrate consists of larger gap sizes or 5′-modified downstream DNA. We have recently shown that Pol β fills small gaps in DNA during microhomology-mediated end-joining as part of a process that increases genomic diversity. Our previous results with single-nucleotide gapped DNA show that Pol β undergoes two pre-catalytic conformational changes upon binding to the correct nucleotide substrate.
The human mitochondrial single-stranded DNA-binding protein displays distinct kinetics and thermodynamics of DNA binding and exchangeThe human mitochondrial ssDNA-binding protein (mtSSB) is a homotetrameric protein, involved in mtDNA replication and maintenance. Although mtSSB is structurally similar to SSB from Escherichia coli (EcoSSB), it lacks the C-terminal disordered domain, and little is known about the biophysics of mtSSB–ssDNA interactions. Here, we characterized the kinetics and thermodynamics of mtSSB binding to ssDNA by equilibrium titrations and stopped-flow kinetic measurements. We show that the mtSSB tetramer can bind to ssDNA in two distinct binding modes: (SSB)30 and (SSB)60, defined by DNA binding site sizes of 30 and 60 nucleotides, respectively.
Kinetic and Structural Impact of Metal Ions and Genetic Variations on Human DNA Polymerase ιDNA polymerase (pol) ι is a Y-family polymerase involved in translesion synthesis, exhibiting higher catalytic activity with Mn2+ than Mg2+. The human germline R96G variant impairs both Mn2+-dependent and Mg2+-dependent activities of pol ι, whereas the Δ1–25 variant selectively enhances its Mg2+-dependent activity. We analyzed pre-steady-state kinetic and structural effects of these two metal ions and genetic variations on pol ι using pol ι core (residues 1–445) proteins. The presence of Mn2+ (0.15 mm) instead of Mg2+ (2 mm) caused a 770-fold increase in efficiency (kpol/Kd,dCTP) of pol ι for dCTP insertion opposite G, mainly due to a 450-fold decrease in Kd,dCTP.
Pre-steady-state Kinetic Analysis of a Family D DNA Polymerase from Thermococcus sp. 9°N Reveals Mechanisms for Archaeal Genomic Replication and MaintenanceBackground: Family D DNA polymerase (polD) is important for replication in most archaea, excluding Crenarchaeota.Results: We report a detailed kinetic characterization of polD nucleotide incorporation, mismatch discrimination, and 3′-5′ exonuclease hydrolysis.Conclusion: Despite evolutionary divergence, polD kinetic pathways share similarities to other DNA polymerase families.Significance: This work contributes to unifying our understanding of DNA polymerase function.
Roles of Residues Arg-61 and Gln-38 of Human DNA Polymerase η in Bypass of Deoxyguanosine and 7,8-Dihydro-8-oxo-2′-deoxyguanosineBackground: Arg-61 and Gln-38 of human DNA polymerase (hpol) η play important roles in the catalytic reaction.Results: Mutations R61M or Q38A/R61A dramatically disrupt the activity of hpol η.Conclusion: Polarized water molecules can mimic and partially compensate for the missing side chains of Arg-61 and Gln-38 in the Q38A/R61A mutant.Significance: The positioning and positive charge of Arg-61 synergistically contribute to the activity of hpol η, with additional effects of Gln-38.