Regulation of re-replication in mammalian cells

Project Summary
Mammalian cells have evolved multiple non-overlapping mechanisms to ensure that DNA replication initiates
from origins of replications once and only once in each division cycle; loss of control over these mechanisms
induces genomic instability, an important driver of malignant transformation. Increasing evidence suggests that
origin utilization and activation in higher eukaryotes is influenced by epigenetic factors, but exact mechanisms
are largely undefined. Our long-term goals are to elucidate the underpinning mechanisms that control replication
initiation in mammalian cells and to understand how perturbations of these mechanisms provokes genomic
instability. The histone methyltransferase SET8 is emerging as a key regulator of replication initiation in
mammalian cells through its mono-methyltransferase activity on histone H4K20. The cell cycle regulated enzyme
is essential for origin licensing in G1 phase of the cell cycle, but is proteolytically degraded in S-phase; blocking
this step triggers reiterative replication initiation within the same cell cycle or re-replication. Both SET8 and
H4K20me, however, are also involved in transcriptional repression and in the repair of DNA double strand breaks
(DSBs), but whether these seemingly independent activities play a role in replication initiation or re-replication is
not known. Most importantly, little to nothing is known about the nature or characteristics of the re-replication
products that accumulate in cells with defective SET8 degradation, nor is there information on where in the
genome re-replication occurs or if certain genomic regions are more prone to re-replication induction. Our new
results show that re-replication resulting from defective SET8 degradation is not a stochastic process, with few
genomic sites exhibit large and significant copy number gains, reminiscent of genomic amplifications that are
seen in cancer cells. Additional preliminary studies suggest that re-replication may originate from DNA double
strand breaks (DSBs) that may spontaneously arise during replication, and requires the activity of genes involved
both in transcriptional silencing and in DSB repair. Our innovative preliminary studies and experimental
approaches are designed to thoroughly examine this alternative model of re-replication induction. In Aim 1, we
will determine the magnitude (copy number gains) and genomic distribution of the re-replicated DNA in bulk and
single cells with defective SET8 degradation and following the induction of DSBs at defined genomic sites. We
will also test if these parameters vary in different cancer cell types and in cancer vs. non-cancer cells. In Aim 2,
we will define the roles of histone H4K20 methylation, transcriptional silencing by the H4K20me reader
L3MBTL1, and proteins involved in the repair of DSBs in effecting re-replication. The successful execution of the
proposed aims promises to increase our understanding of the mechanisms regulating replication initiation in
mammalian cells, and lead to a better understanding of how perturbations of these mechanisms provokes
genomic instability.

Grant Number: 5R01GM135376-05
NIH Institute/Center: NIH
Principal Investigator: TAREK ABBAS