Mechanistic insight into genome stability pathways

Project Summary
Genome integrity depends on a robust DNA replication program and the activity of replication-coupled repair
pathways that operate during different phases of the cell cycle. My laboratory has had a longstanding interest in
the causes and consequences of replication stress. Replication stress arises when lesions in the genome persist
due to repair deficiencies or when components of the replication machinery are dysfunctional. Although disease-
causing mutations in essential replication factors are rare, they can cause pleiotropic and severe disorders, such
as immunodeficiency, cardiomyopathy, or growth defects. In recent years, we have investigated the molecular
mechanism that underlies these rare diseases. We have identified compound heterozygous patient mutations in
the replication factor minichromosome maintenance protein 10 (MCM10), and have modeled them in human
somatic cell lines. Although these mutations cause relatively mild cellular replication defects, they pose
significant problems to telomere maintenance. One caveat of the current cell models is that they are immortalized
and express telomerase constitutively. To better understand the impact of replication defects in the context of
cellular development of affected tissues, we propose to engineer genome-edited induced pluripotent stem cells
and differentiate them into specific cell types in vitro. This presents a valuable alternative to animal models which,
relevantly, do not fully mimic telomere homeostasis in humans. Moreover, we are interested in the pathways that
cells activate for survival under conditions of mild replication stress. Previous work has identified a network based
on ubiquitination and SUMOylation, and ring finger protein 4 (RNF4) as a key component. RNF4 is a SUMO-
targeted E3 ubiquitin ligase that has been implicated in double-strand break repair, however, its role at replication
forks and in telomere maintenance is not well understood. A genetic interaction screen has identified Bloom
helicase (BLM), a RecQ-family helicase that causes premature aging, and ubiquitin specific peptidase 7 (USP7),
a deubiquitinase, as strong negative interactors. Mutations in USP7 have been linked to rare
neurodevelopmental disorders, but its cellular action has remained obscure. Interestingly, USP7 and BLM also
regulate DNA replication and telomere length. We will investigate the relationship between RNF4, USP7 and
BLM in chromosome inheritance in telomerase-positive and -negative cells. Lastly, a common feature of
replication stress is under-replication due to an inability to duplicate the entire genome. As a result, single-
stranded gaps persist that can either be filled by post-replicative repair that is regulated by the ubiquitination of
PCNA or, as a last resort, by mitotic DNA synthesis (MiDAS). MiDAS is a break-induced replication (BIR)-like
pathway that, unlike a classical replication fork, copies DNA by displacement synthesis. We will study how
ubiquitinated PCNA controls MiDAS, and will determine whether other BIR-related pathways are regulated by
PCNA ubiquitination. In summary, the questions addressed in this proposal will elucidate fundamental and
disease-relevant mechanisms of genome stability pathways in human cells.

Grant Number: 5R35GM141805-06
NIH Institute/Center: NIH
Principal Investigator: Anja-Katrin Bielinsky