Student Theses and Dissertations

Date of Award

2020

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Smogorzewska Laboratory

Abstract

Proteasome shuttle proteins, DNA Damage Inducible 1 (DDI1) and DNA Damage Inducible 2 (DDI2), are active in the replication stress response (Kottemann et al., 2018). Under conditions of replication stress, DDI1/2 function to remove Replication Termination Factor 2 (RTF2) from the replisome. Persistence of RTF2 at the replisome compromises the replication stress response, resulting in accumulation of single-stranded DNA, inefficient replication fork restart, genome instability, and cell death. During unperturbed replication, RTF2 travels with the replication fork (Kottemann et al., 2018). Here we examine the function of RTF2 and address why RTF2’s removal from the replisome is necessary for recovery from replication stress. We find that loss of RTF2 results in murine embryonic lethality and that RTF2 is required for normal DNA replication elongation speeds. By isolation of proteins on nascent DNA (iPOND), replisomes deficient for RTF2 concurrently lack RNase H2, a heterotrimeric enzyme responsible for removing RNA in the context of RNA-DNA heteroduplexes. Like RTF2, RNase H2 is necessary for normal replication speed. RTF2- deficient cells exhibit phenotypes consistent with loss of RNase H2 and an increase in genome-embedded ribonucleotides, including the accumulation of DNA damage. In the absence of DDI1/2, the persistence of RNase H2 at a stalled replication fork also compromises the replication stress response. We show that direct replication restart is dependent on PRIM1, the primase component of DNA polymerase α. The dependence on PRIM1 for replication restart is greater than its requirement during processive DNA replication. Our data suggest there might be a competition between PRIM1 and RNase H2, which regulates the synthesis and degradation of the RNA primer. Our data support a model whereby the DDI-RTF2 axis regulates RNase H2 levels at the replisome during stress. RNase H2 travels with the replication fork to remove genome-embedded ribonucleotides resulting from random polymerase incorporation or inefficient Okazaki fragment maturation. However, upon DNA damage and replication stalling, RNase H2 must be removed from the replication fork to allow for proper restart using PRIM1-synthesized RNA primers. The work presented here provides a new perspective on the reorganization of the replisome that is required upon replication stress and addresses a fundamental need for regulation of replication-coupled ribonucleotide incorporation during DNA replication and repair.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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