Interlocking activities of DNA polymerase β in the base excision repair pathway.

SignificanceBase excision repair (BER) is one of the major DNA repair pathways used to fix a myriad of cellular DNA lesions. The enzymes involved in BER, including DNA polymerase β (Polβ), have been identified and characterized, but how they act together to efficiently perform BER has not been fully understood. Through gel electrophoresis, mass spectrometry, and kinetic analysis, we discovered that the two enzymatic activities of Polβ can be interlocked, rather than functioning independently from each other, when processing DNA intermediates formed in BER.

Optimization of proteomics sample preparation for forensic analysis of skin samples.

We present an efficient protein extraction and in-solution enzymatic digestion protocol optimized for mass spectrometry-based proteomics studies of human skin samples. Human skin cells are a proteinaceous matrix that can enable forensic identification of individuals. We performed a systematic optimization of proteomic sample preparation for a protein-based human forensic identification application.

Fractionation of DNA and protein from individual latent fingerprints for forensic analysis.

Human touch samples represent a significant portion of forensic DNA casework. Yet, the generally low abundance of genetic material combined with the predominantly extracellular nature of DNA in these samples makes DNA-based forensic analysis exceptionally challenging. Human proteins present in these same touch samples offer an abundant and environmentally-robust alternative.

An algorithm for random match probability calculation from peptide sequences.

For the past three decades, forensic genetic investigations have focused on elucidating DNA signatures. While DNA has a number of desirable properties (e.g.

Temporal Dynamics of the Soil Metabolome and Microbiome During Simulated Anaerobic Soil Disinfestation.

Significant interest exists in engineering the soil microbiome to attain suppression of soil-borne plant diseases. Anaerobic soil disinfestation (ASD) has potential as a biologically regulated disease control method; however, the role of specific metabolites and microbial community dynamics contributing to ASD mediated disease control is mostly uncharacterized. Understanding the trajectory of co-evolutionary processes leading to syntrophic generation of functional metabolites during ASD is a necessary prelude to the predictive utilization of this disease management approach.

Kinetic Mechanism of DNA Polymerases: Contributions of Conformational Dynamics and a Third Divalent Metal Ion.

Faithful transmission and maintenance of genetic material is primarily fulfilled by DNA polymerases. During DNA replication, these enzymes catalyze incorporation of deoxynucleotides into a DNA primer strand based on Watson-Crick complementarity to the DNA template strand. Through the years, research on DNA polymerases from every family and reverse transcriptases has revealed structural and functional similarities, including a conserved domain architecture and purported two-metal-ion mechanism for nucleotidyltransfer.

Time-Dependent Extension from an 8-Oxoguanine Lesion by Human DNA Polymerase Beta.

The oxidative DNA lesion 7,8-dihydro-2'-deoxyguanine (8-oxoG) often occurs in double-stranded DNA and poses a threat to genomic integrity due to the ability of 8-oxoG to form stable Watson-Crick base pairs with deoxycytidine (8-oxoG:dC) and Hoogsteen base pairs with deoxyadenosine (8-oxoG:dA). In humans, short-patch base excision repair of 8-oxoG:dA base pairs requires human DNA polymerase β (hPolβ) to bypass 8-oxoG. Previously, we have shown hPolβ-catalyzed 8-oxoG bypass to exhibit low fidelity and identified a unique stacking interaction between the newly incorporated nucleotide (dCMP or dAMP) and the templating 8-oxoG.

Structural basis for the D-stereoselectivity of human DNA polymerase β.

Nucleoside reverse transcriptase inhibitors (NRTIs) with L-stereochemistry have long been an effective treatment for viral infections because of the strong D-stereoselectivity exhibited by human DNA polymerases relative to viral reverse transcriptases. The D-stereoselectivity of DNA polymerases has only recently been explored structurally and all three DNA polymerases studied to date have demonstrated unique stereochemical selection mechanisms. Here, we have solved structures of human DNA polymerase β (hPolβ), in complex with single-nucleotide gapped DNA and L-nucleotides and performed pre-steady-state kinetic analysis to determine the D-stereoselectivity mechanism of hPolβ.

Advances in Structural and Single-Molecule Methods for Investigating DNA Lesion Bypass and Repair Polymerases.

Innovative advances in X-ray crystallography and single-molecule biophysics have yielded unprecedented insight into the mechanisms of DNA lesion bypass and damage repair. Time-dependent X-ray crystallography has been successfully applied to view the bypass of 8-oxo-7,8-dihydro-2'-deoxyguanine (8-oxoG), a major oxidative DNA lesion, and the incorporation of the triphosphate form, 8-oxo-dGTP, catalyzed by human DNA polymerase β. Significant findings of these studies are highlighted here, and their contributions to the current mechanistic understanding of mutagenic translesion DNA synthesis (TLS) and base excision repair are discussed.

Structural Insights into the Post-Chemistry Steps of Nucleotide Incorporation Catalyzed by a DNA Polymerase.

DNA polymerases are essential enzymes that faithfully and efficiently replicate genomic information.1-3 The mechanism of nucleotide incorporation by DNA polymerases has been extensively studied structurally and kinetically, but several key steps following phosphodiester bond formation remain structurally uncharacterized due to utilization of natural nucleotides. It is thought that the release of pyrophosphate (PP) triggers reverse conformational changes in a polymerase in order to complete a full catalytic cycle as well as prepare for DNA translocation and subsequent incorporation events.

Viewing Human DNA Polymerase β Faithfully and Unfaithfully Bypass an Oxidative Lesion by Time-Dependent Crystallography.

One common oxidative DNA lesion, 8-oxo-7,8-dihydro-2'-deoxyguanine (8-oxoG), is highly mutagenic in vivo due to its anti-conformation forming a Watson-Crick base pair with correct deoxycytidine 5'-triphosphate (dCTP) and its syn-conformation forming a Hoogsteen base pair with incorrect deoxyadenosine 5'-triphosphate (dATP). Here, we utilized time-resolved X-ray crystallography to follow 8-oxoG bypass by human DNA polymerase β (hPolβ). In the 12 solved structures, both Watson-Crick (anti-8-oxoG:anti-dCTP) and Hoogsteen (syn-8-oxoG:anti-dATP) base pairing were clearly visible and were maintained throughout the chemical reaction.

Vertical distribution and diversity of bacteria and archaea in sulfide and methane-rich cold seep sediments located at the base of the Florida Escarpment.

The bacterial and archaeal communities of the sediments at the base of the Florida Escarpment (Gulf of Mexico, USA) were investigated using molecular phylogenetic analysis. The total microbial community DNA of each of three vertical zones (top, middle and bottom) of a sediment core was extracted and the 16S rRNA genes were amplified by PCR, cloned and sequenced. Shannon-Weaver Diversity measures of bacteria were high in all three zones.