Srs2 promotes synthesis-dependent strand annealing by disrupting DNA polymerase δ-extending D-loops.

Synthesis-dependent strand annealing (SDSA) is the preferred mode of homologous recombination in somatic cells leading to an obligatory non-crossover outcome, thus avoiding the potential for chromosomal rearrangements and loss of heterozygosity. Genetic analysis identified the Srs2 helicase as a prime candidate to promote SDSA. Here, we demonstrate that Srs2 disrupts D-loops in an ATP-dependent fashion and with a distinct polarity. Specifically, we partly reconstitute the SDSA pathway using Rad51, Rad54, RPA, RFC, DNA Polymerase δ with different forms of PCNA. Consistent with genetic data showing the requirement for SUMO and PCNA binding for the SDSA role of Srs2, Srs2 displays a slight but significant preference to disrupt extending D-loops over unextended D-loops when SUMOylated PCNA is present, compared to unmodified PCNA or monoubiquitinated PCNA. Our data establish a biochemical mechanism for the role of Srs2 in crossover suppression by promoting SDSA through disruption of extended D-loops

Revealing the role of the product metal in DNA polymerase β catalysis

DNA polymerases catalyze a metal-dependent nucleotidyl transferase reaction during extension of a DNA strand using the complementary strand as a template. The reaction has long been considered to require two magnesium ions. Recently, a third active site magnesium ion was identified in some DNA polymerase product crystallographic structures, but its role is not known. Using quantum mechanical/ molecular mechanical calculations of polymerase β, we find that a third magnesium ion positioned near the newly identified product metal site does not alter the activation barrier for the chemical reaction indicating that it does not have a role in the forward reaction. This is consistent with time-lapse crystallographic structures following insertion of Sp-dCTPαS. Although sulfur substitution deters product metal binding, this has only a minimal effect on the rate of the forward reaction. Surprisingly, monovalent sodium or ammonium ions, positioned in the product metal site, lowered the activation barrier. These calculations highlight the impact that an active site water network can have on the energetics of the forward reaction and how metals or enzyme side chains may interact with the network to modulate the reaction barrier. These results also are discussed in the context of earlier findings indicating that magnesium at the product metal position blocks the reverse pyrophosphorolysis reaction

Specific mutations in the D1–D2 linker region of VCP/p97 enhance ATPase activity and confer resistance to VCP inhibitors

A grant from the One-University Open Access Fund at the University of Kansas was used to defray the author's publication fees in this Open Access journal. The Open Access Fund, administered by librarians from the KU, KU Law, and KUMC libraries, is made possible by contributions from the offices of KU Provost, KU Vice Chancellor for Research & Graduate Studies, and KUMC Vice Chancellor for Research. For more information about the Open Access Fund, please see http://library.kumc.edu/authors-fund.xml.Valosin-containing protein (VCP), together with several partner proteins, extracts ubiquitinated client proteins from E3 ligase complex and facilitates their degradation through ubiquitin–proteasome system. Therefore, it plays an important role in regulating protein quality control and various cellular pathways. Recent studies also identified VCP as a lineage-specific essential gene in ovarian cancer. An orally bioavailable VCP inhibitor, CB-5083, is currently in Phase I clinical trials because it shows therapeutic effects in multiple tumor xenograft models. However, the mechanism of resistance to CB-5083 is unknown. Here, we characterized molecular mechanism of resistance to CB-5083. Using incremental exposure to CB-5083, we established CB-5083-resistant ovarian cancer cells that showed five- to six-fold resistance in vitro compared with parental cells. Genomic and complementary DNA sequencing of the VCP coding region revealed a pattern of co-selected mutations: (1) missense mutations at codon 470 in one copy resulting in increased ATPase activity and (2) nonsense or frameshift mutations at codon 606 or codon 616 in another copy causing the loss of allele-specific expression. Unbiased molecular docking studies showed codon 470 as a putative binding site for CB-5083. Furthermore, the analysis of somatic mutations in cancer genomes from the Cancer Genome Atlas (TCGA) indicated that codon 616 contains hotspot mutations in VCP. Thus, identification of these mutations associated with in vitro resistance to VCP inhibitors may be useful as potential theranostic markers while screening for patients to enroll in clinical trials. VCP has emerged as a viable therapeutic target for several cancer types, and therefore targeting such hyperactive VCP mutants should aid in improving the therapeutic outcome in cancer patients

Requirement for transient metal ions revealed through computational analysis for DNA polymerase going in reverse

DNA polymerases use a general two-metal ion mechanism for DNA synthesis. Recent time-lapse crystallographic studies identified additional adjunct metal ions in the polymerase active site. One of these ions correlates with appearance of pyrophosphate and was proposed to be involved in pyrophosphorolysis (reverse reaction of DNA synthesis). Because DNA polymerases can use pyrophosphorolysis to remove chain-terminating nucleotides during chemotherapies, a better understanding of this reaction is warranted. Through site-directed mutagenesis, pyrophosphorolysis measurements, and computational analysis, we examine the role of metal ions in the reverse reaction. The results indicate that the product-associated metal ion facilitates pyrophosphorolysis during the early stages of the reaction but deters the reaction at later stages, suggesting dynamic metal behavior that can modulate the chemical equilibrium

A charged residue at the subunit interface of PCNA promotes trimer formation by destabilizing alternate subunit interactions

Eukaryotic proliferating cell nuclear antigen (PCNA), an essential accessory factor in DNA replication and repair, is a ring-shaped homotrimer. A novel nontrimeric structure of E113G-mutant PCNA protein is reported, which shows that this protein forms alternate subunit interactions. It is concluded that the charged side chain of Glu113 promotes normal trimer formation by destabilizing these alternate subunit interactions

Mechanisms of telomerase inhibition by oxidized and therapeutic dNTPs

Telomerase enzymes add telomeric repeats to the end of linear chromosomes. Here the authors reveal mechanisms by which oxidized dNTPs and therapeutic dNTPs inhibit telomerase-mediated telomere elongation

Altered APE1 activity on abasic ribonucleotides is mediated by changes in the nucleoside sugar pucker

Ribonucleotides (rNTPs) are predicted to be incorporated into the genome at a rate of up to 3 million times per cell division, making rNTPs the most common non-standard nucleotide in the human genome. Typically, misinserted ribonucleotides are repaired by the ribonucleotide excision repair (RER) pathway, which is initiated by RNase H2 cleavage. However, rNTPs are susceptible to spontaneous depurination generating abasic ribonucleotides (rAPs), which are unable to be processed by RNase H2. Additionally, rAPs have been found in nascent RNA and coupled to R-loops. Recent work identified that base excision repair (BER) protein AP-Endonuclease 1 (APE1) is responsible for the initial processing of rAPs embedded in DNA and in R-loops. APE1 is a well characterized AP endonuclease that cleaves 5′ of abasic sites, but its ability to cleave at rAPs remains poorly understood. Here, we utilize enzyme kinetics, X-ray crystallography, and molecular dynamics simulations to provide insight into rAP processing by APE1. Enzyme kinetics were used to determine pre-steady-state rates of APE1 cleavage on DNA substrates containing rAP, revealing a decrease in activity compared to cleavage at a canonical deoxy-AP substrate. Using X-ray crystallography, we identified novel contacts between the rAP and the APE1 active site. We demonstrate that the rAP sugar pucker is accommodated in the active site in a C3′-endo conformation, influencing its position and contributing to a decrease in activity compared to the deoxy-AP site. Together, this work provides molecular level insights into rAP processing by APE1 and advances our understanding of ribonucleotide processing within genomic DNA

Altered Nucleotide Insertion Mechanisms of Disease-Associated TERT Variants

Telomere biology disorders (TBDs) are a spectrum of diseases that arise from mutations in genes responsible for maintaining telomere integrity. Human telomerase reverse transcriptase (hTERT) adds nucleotides to chromosome ends and is frequently mutated in individuals with TBDs. Previous studies have provided insight into how relative changes in hTERT activity can lead to pathological outcomes. However, the underlying mechanisms describing how disease-associated variants alter the physicochemical steps of nucleotide insertion remain poorly understood. To address this, we applied single-turnover kinetics and computer simulations to the Tribolium castaneum TERT (tcTERT) model system and characterized the nucleotide insertion mechanisms of six disease-associated variants. Each variant had distinct consequences on tcTERT’s nucleotide insertion mechanism, including changes in nucleotide binding affinity, rates of catalysis, or ribonucleotide selectivity. Our computer simulations provide insight into how each variant disrupts active site organization, such as suboptimal positioning of active site residues, destabilization of the DNA 3′ terminus, or changes in nucleotide sugar pucker. Collectively, this work provides a holistic characterization of the nucleotide insertion mechanisms for multiple disease-associated TERT variants and identifies additional functions of key active site residues during nucleotide insertion