The genome-wide analysis of the specification and activation of human chromosomal DNA replication origin sites

Autor: Wilkes, Helen Sarah
Rok vydání: 2021
Předmět:
DOI: 10.17863/cam.72547
Popis: Background: Human DNA replication is a critical cellular process that initiates at tens of thousands of DNA replication origins. Next-generation sequencing (NGS) enabled the elucidation of some characteristics that define human replication origins. Initiation-site sequencing (iniSeq) utilises the established human cell-free system for in vitro DNA replication, whereby a digoxigenin tag is introduced into newly synthesised DNA. Newly synthesised DNA was separated from total genomic DNA by immunoprecipitation and subjected to NGS. Additionally, the replication factors Y RNAs and Xenopus laevis Nucleosome Remodelling and histone Deacetylation (xNuRD) complex are essential for DNA replication initiation, but the mechanism(s) involved remain unelucidated. The precise mechanisms of DNA replication, and factors involved in human replication origin specification and activation are not fully known. My work: I developed and improved upon iniSeq to produce a novel DNA replication origin NGS method. Density-substitution initiation-site sequencing (ds-iniSeq) exploited the semi-conservative nature of replicated DNA to identify human replication origins and determine their relative activities. During a short in vitro replication reaction, newly synthesised DNA incorporated a heavy nucleotide (BrdUTP) which resulted in Heavy-Light replicated and Light-Light unreplicated DNA. This density substitution enabled reliable separation of replicated and unreplicated DNA, which underwent NGS and processing by MACS peak calling to identify the origins; Heavy-Light DNA: Light-Light DNA ratios at identified replication origins determined their relative activities. I identified ~14,000 discrete replication origins and showed that CpG islands and CpG island-promoters were dominant features associated with high origin activity. Origins identified by ds-iniSeq (ds-iniSeq origins) strongly colocalised with transcription start sites and active genes, but transcriptional activity did not correlate with origin activity. Ds-iniSeq origins colocalised with previously identified early firing origin-associated histone marks. I employed ds-iniSeq to examine how Y RNAs and xNuRD impact replication origin specification and activation genome-wide, by manipulating the in vitro replication reactions. Y RNA removal resulted in fewer ds-iniSeq origins with lower activities genome-wide. xNuRD restored the number and activities of origins genome-wide to similar levels when in the presence of Y RNAs. I adapted ds-iniSeq to assess replication elongation; density-substitution elongation-site sequencing (ds-eloSeq) had a longer replication reaction incubation. Conducting ds-eloSeq alongside ds-iniSeq enabled the assessment of the transition from replication initiation to elongation, and the impact of replication factors on this relationship. I performed a preliminary analysis of the impact of Y RNA removal and xNuRD addition on elongation which revealed reduced site activities of DNA regions adjacent to the corresponding ds-iniSeq origins in the absence of Y RNAs. I have also endeavoured to develop a new method for calling replicating ds-eloSeq sites as MACS was inappropriate for these samples. Finally, Y RNAs bind to various proteins during DNA replication. From mass spectrometric data (generated by M.Kowalski), I identified the Polycomb repressive complex 2 (PRC2) as a Y RNA binding complex that possessed DNA replication activity (in the human cell-free system). I showed that PRC2 plays a stimulatory role in DNA replication initiation. In summary, I have established a novel NGS method for origin identification that determines relative replication origin activity and used it to assess the impact of genomic and epigenetic features, and hY RNAs and xNuRD, on origin specification and activation. I have further developed a NGS method to examine elongation and, using biochemical techniques, have characterised PRC2 as a stimulatory DNA replication initiation factor.
St Catharine's College, Cambridge Zoology Department, Cambridge
Databáze: OpenAIRE
Externí odkaz: