Acetylation of DNA Polymerase Beta Regulates the Choice of the Base Excision Repair Pathway

poster abstractBase excision repair (BER) is the main pathway through which base damages are repaired in the cell. Single nucleotide damage can be corrected either through short patch BER (SP-BER), in which the single damaged base is replaced, or long patch BER (LP-BER), in which two or more nucleotides can be replaced. Several proteins are involved in the process including DNA polymerase beta (pol β) and FEN1, both of which are the focus for this study. DNA pol β is a multifunctional protein which contains both polymerase and lyase properties. In LP-BER, pol β displaces the uncleaved 5’dRP moiety into a flap structure which is recognized and cleaved by FEN1 and subsequently ligated by DNA ligase 1. Previous in vitro studies show that pol β acetylation reduces lyase activity, requiring repair to proceed via LP-BER. In this study, we determined the effect of in vitro acetylation on the enzymatic activities of DNA pol β and FEN1. Both unmodified and acetylated forms of pol β were tested for their synthesis and strand displacement activities. Interestingly, acetylated forms of pol β showed much greater activity at all concentrations versus unmodified forms. Interestingly we also found that FEN1 cleavage activity was increased in reactions containing acetylated pol β compared to the unmodified form due to the increased strand displacement activity of the polymerase. Our results suggest that the acetylated form of DNA pol β more actively participates in LP-BER, creating longer strands of corrected, higher fidelity nucleotides

Understanding the Efficacy of Inhibitor TRDL-551 on Replication Protein A (RPA) Binding Affinity

poster abstractReplication protein A (RPA), a single stranded DNA (ssDNA) binding protein, prevents ssDNA from being a target of cellular nucleases, recombining with other complementary sequences, or folding-back on itself to form hairpin structures. Each of these scenarios can cause high rates of genome stability. Due to the protective function of RPA it now used as a therapeutic target for cancer cells, wherein DNA replication and repair often occur at high frequencies. For the current study, we utilized a chemical inhibitor of RPA, TDRL-551, which has been previously characterized as a potent inhibitor of RPA binding to substrate. In this study, we chose to further analyze the TDRL-551 inhibitor by testing different binding conditions either by the unmodified or acetylated form of RPA. Results from our laboratory have shown that acetylated RPA binds with higher affinity to ssDNA compared to the unmodified form. We tested RPA (unmodified and acetylated) binding under two different conditions; (i) RPA was pre-bound to substrate and then incubated with inhibitor or (ii) RPA was pre-bound to inhibitor and then exposed to substrate. Our results showed that in the first case, there was no effect of exposure to inhibitor on both unmodified and acetylated RPA binding. However, when RPA was first incubated with the inhibitor, both the unmodified and acetylated RPA showed reduced binding affinity to the ssDNA substrate. In conclusion, our studies show that the length of the DNA substrate, the posttranslational status of RPA and the time of exposure of the inhibitor to the RPA all play a significant role in determining the potency of this inhibitor. Further studies are being done to ascertain the efficacy of this inhibitor before using it in translational research

Dual agarose magnetic (DAM) ChIP

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HDAC inhibitors stimulate viral transcription by multiple mechanisms

<p>Abstract</p> <p>Background</p> <p>The effects of histone deacetylase inhibitor (HDACi) treatment on SV40 transcription and replication were determined by monitoring the levels of early and late expression, the extent of replication, and the percentage of SV40 minichromosomes capable of transcription and replication following treatment with sodium butyrate (NaBu) and trichostatin A (TSA).</p> <p>Results</p> <p>The HDACi treatment was found to maximally stimulate early transcription at early times and late transcription at late times through increased numbers of minichromosomes which carry RNA polymerase II (RNAPII) transcription complexes and increased occupancy of the transcribing minichromosomes by RNAPII. HDACi treatment also partially relieved the normal down-regulation of early transcription by T-antigen seen later in infection. The increased recruitment of transcribing minichromosomes at late times was correlated to a corresponding reduction in SV40 replication and the percentage of minichromosomes capable of replication.</p> <p>Conclusion</p> <p>These results suggest that histone deacetylation plays a critical role in the regulation of many aspects of an SV40 lytic infection.</p

Epigenetic Analysis of SV40 Minichromosomes

Simian virus 40 (SV40) is one of the best-characterized members of the polyomavirus family of small DNA tumor viruses. It has a small genome of 5243 bp and utilizes cellular proteins for its molecular biology, with the exception of the T-antigen protein, which is coded by the virus and is involved in regulating transcription and directing replication. Importantly, SV40 exists as chromatin in both the virus particle and intracellular minichromosomes. These facts, combined with high yields of virus and minichromosomes following infection and ease of manipulation, have made SV40 an extremely useful model to study all aspects of eukaryotic molecular biology. This unit describes procedures for working with SV40 and preparing SV40 chromatin from infected cells and virus particles, as well as procedures for using SV40 chromatin to study epigenetic regulation

Acetylation of Replication Protein A (RPA) Improves its DNA Binding Property

poster abstractGenome maintenance is critical for cellular survival and growth. Replication Protein A (RPA), a single-strand DNA (ssDNA) binding protein, is vital for various aspects of genome maintenance such as replication, recombination, repair and checkpoint activation. RPA binding to ssDNA protects it from degradation by cellular nucleases, prevents secondary structure formation and from illegitimate recombination. Within the cell, RPA is subject to many post-translational modifications including phosphorylation, SUMOylation and ribosylation. These modifications regulate the activity of RPA with DNA and other binding partners. RPA has been reported to be also modified by acetylation. We found that human RPA (hRPA) can be in vitro acetylated by p300, an acetyl transferase (AT). To study the effect of this modification on its ssDNA binding function, we made use of electro-mobility gel shift assay (EMSA) and bio-layer interferometry (BLI) technology. Using various length oligos, we tested the binding property of unmodified and acetylated RPA. Our results showed that acetylation of RPA increased its binding affinity compared to unmodified RPA. Interestingly, the acetylated form was also able to bind more stably to shorter length oligos compared to the unmodified form. This suggests that the acetylation of RPA improves its ssDNA binding function. This alteration in its enzymatic activity would have significant implications in maintenance of genome fidelity since improved DNA binding function of RPA will protect the genome from both endogenous and exogenous stresses. Additionally, using mass spectrometry analysis we have identified the lysine residues that get modified by the acetyl group both in vitro and in vivo. We are currently studying the factors that trigger this post-translational modification in the cell

HMGB1 Stimulates Activity of Polymerase β on Nucleosome Substrates

The process of base excision repair (BER) recognizes and repairs small lesions or inappropriate bases on DNA through either a short-patch or long-patch pathway. The enzymes involved in BER have been well-characterized on DNA substrates, and, somewhat surprisingly, many of these enzymes, including several DNA glycosylases, AP endonuclease (APE), FEN1 endonuclease, and DNA ligases, have been shown to have activity on DNA substrates within nucleosomes. DNA polymerase β (Pol β), however, exhibits drastically reduced or no activity on nucleosomal DNA. Interestingly, acetylation of Pol β, by the acetyltransferase p300, inhibits its 5′ dRP-lyase activity and presumably pushes repair of DNA substrates through the long-patch base excision repair (LP-BER) pathway. In addition to the major enzymes involved in BER, a chromatin architectural factor, HMGB1, was found to directly interact with and enhance the activity of APE1 and FEN1, and thus may aid in altering the structure of the nucleosome to be more accessible to BER factors. In this work, we investigated whether acetylation of Pol β, either alone or in conjunction with HMGB1, facilitates its activity on nucleosome substrates. We find acetylated Pol β exhibits enhanced strand displacement synthesis activity on DNA substrates, but, similar to the unmodified enzyme, has little or no activity on nucleosomes. Preincubation of DNA templates with HMGB1 has little or no stimulatory effect on Pol β and even is inhibitory at higher concentrations. In contrast, preincubation of nucleosomes with HMGB1 rescues Pol β gap-filling activity in nucleosomes, suggesting that this factor may help overcome the repressive effects of chromatin

Biochemical analyses indicate that binding and cleavage specificities define the ordered processing of human Okazaki fragments by Dna2 and FEN1

In eukaryotic Okazaki fragment processing, the RNA primer is displaced into a single-stranded flap prior to removal. Evidence suggests that some flaps become long before they are cleaved, and that this cleavage involves the sequential action of two nucleases. Strand displacement characteristics of the polymerase show that a short gap precedes the flap during synthesis. Using biochemical techniques, binding and cleavage assays presented here indicate that when the flap is ∼30 nt long the nuclease Dna2 can bind with high affinity to the flap and downstream double strand and begin cleavage. When the polymerase idles or dissociates the Dna2 can reorient for additional contacts with the upstream primer region, allowing the nuclease to remain stably bound as the flap is further shortened. The DNA can then equilibrate to a double flap that can bind Dna2 and flap endonuclease (FEN1) simultaneously. When Dna2 shortens the flap even more, FEN1 can displace the Dna2 and cleave at the flap base to make a nick for ligation

The 9-1-1 checkpoint clamp stimulates DNA resection by Dna2-Sgs1 and Exo1

Single-stranded DNA (ssDNA) at DNA ends is an important regulator of the DNA damage response. Resection, the generation of ssDNA, affects DNA damage checkpoint activation, DNA repair pathway choice, ssDNA-associated mutation and replication fork stability. In eukaryotes, extensive DNA resection requires the nuclease Exo1 and nuclease/helicase pair: Dna2 and Sgs1^(BLM). How Exo1 and Dna2-Sgs1^(BLM) coordinate during resection remains poorly understood. The DNA damage checkpoint clamp (the 9-1-1 complex) has been reported to play an important role in stimulating resection but the exact mechanism remains unclear. Here we show that the human 9-1-1 complex enhances the cleavage of DNA by both DNA2 and EXO1 in vitro, showing that the resection-stimulatory role of the 9-1-1 complex is direct. We also show that in Saccharomyces cerevisiae, the 9-1-1 complex promotes both Dna2-Sgs1 and Exo1-dependent resection in response to uncapped telomeres. Our results suggest that the 9-1-1 complex facilitates resection by recruiting both Dna2-Sgs1 and Exo1 to sites of resection. This activity of the 9-1-1 complex in supporting resection is strongly inhibited by the checkpoint adaptor Rad9^(53BP1). Our results provide important mechanistic insights into how DNA resection is regulated by checkpoint proteins and have implications for genome stability in eukaryotes