Study on DNA damage response and chromatin dynamics using biochemical and biophysical techniques

Abstract

DNA must be preserved without damage to maintain genomic stability. If damaged DNA is not repaired properly and accumulates, genome integrity is disrupted, posing a threat to the organism's maintenance. Therefore, it is important for to comprehending DNA damage repair for the preservation of genome homeostasis. Additionally, since eukaryotic DNA is wrapped around proteins called histones to form a compacted structure called chromatin, it is imperative to investigate the mechanism of chromatin dynamics to understand the DNA repair processes that occur within cells. I have studied DNA damage repair and chromatin dynamics caused by histone chaperones. Through these studies, I will provide an insight to understand the link between gene damage repair and epigenetics. Chapter 1 Homologous recombination (HR) mediated DNA double strand break (DSB) repair is initiated by the end resection. DSB repair pathway is dependent on the extent of DNA end resection. Although various nucleases for end resection have been studied, the detailed mechanism how the initial nicked DNA generated by MRE11 RAD50 NBS1 is recognized and subsequent nucleases are recruited to DSB sites to lead end resection is still elusive. I investigated that the MSH2 MSH3 mismatch repair complex is recruited to DSB sites and recognizes the initial nicked DNA by interacting with the chromatin remodeling protein SMARCAD1. MSH2 MSH3 promotes to recruit EXO1 for long range resection and enhances its catalytic activity. Moreover, MSH2 MSH3 prevents the access of POLQ, which promotes polymerase theta mediated end joining (TMEJ). Taken together, I suggest a direct role of MSH2 MSH3 in the initiation steps of DSB repair by enhancing end resection and affecting DSB repair pathway by favoring HR over TMEJ.

Chapter 2 DNA replication is crucial for maintaining genomic stability and cell survival. During this process, replication stress can be induced by shortage of replication resources or topological obstacles for replication. In the presence of DNA replication stress, RPA, a eukaryotic single-stranded DNA (ssDNA) binding protein, is phosphorylated by the ataxia telangiectasia and RAD3-related (ATR) kinase at the early stage, which initiates DNA damage response. Recently, it was reported that NSMF (N -methyl-D-aspartate receptor synaptonuclear signaling and neuronal migration factor), which is related to the neuronal plasticity and development, promotes RPA32 phosphorylation via ATR upon replication stress. I discovered that NSMF selectively displaces the more-weakly bound 8- and 20-nt binding modes of RPA from ssDNA allowing the retention of the more stable RPA molecules in the 30-nt binding mode. I further demonstrated that the 30-nt mode of RPA enhances RPA32 phosphorylation by ATR and that the phosphorylated RPA becomes stabilized on ssDNA. This research suggests that the stable binding mode of RPA enhances RPA32 phosphorylation by ATR under the replication stress and gives insight into a new role of NSMF in ATR pathway.

Chapter 3 Chromatin assembly and disassembly during DNA metabolic events are important for genome maintenance and epigenetic inheritance. Nucleosomes are assembled or disassembled in an ATP-independent manner by histone chaperones such as CAF-1 and FACT. It was recently reported that Abo1, a bromodomain-containing AAA+ ATPase of fission yeast, plays a role in chromatin assembly. However, the biological roles and molecular features of Abo1 remain poorly understood. I characterized the molecular function of Abo1 as a histone chaperone. Using a novel single-molecule imaging technique called DNA curtain, I demonstrated that Abo1 mediates to load H3-H4 dimers onto DNA. I also showed that the histone loading by Abo1 requires ATP hydrolysis. This study provides that Abo1 is a novel histone chaperone that can mediate to load histone H3-H4 onto DNA via ATP hydrolysis.