EMBO Fellows Meeting 2012

Mong Sing Lai
EMBO Fellows Meeting 2012
Mechanisms controlling terminal fork integrity and replicon dynamics following
double strand break formation
Abstract
The understanding of the molecular mechanisms allowing cell survival in response to replication stress is
important to elucidate those processes that protect the integrity of replicating chromosomes following
oncogenic insults. In response to replication stress, Mec1/ATR-dependent checkpoint response and
specialized SUMO/Ubiquitin pathways control the stalled and damaged replication fork stability. By contrast,
Tel1/ATM-dependent checkpoint response and MRX/MRN complex protect the integrity of replication forks
collapsing at the double strand break (DSB) sites (termed the terminal fork) preventing abnormal transitions.
Recent data further suggest that Mec1/ATR-dependent checkpoint response controls the physical connections
between replicating chromosomes and the nuclear envelope to facilitate fork progression across transcribed
units and to prevent aberrant topological transitions at stalled replication forks. Using a combination of
mechanistic and genomic approaches in budding yeast, we previously shown that terminal forks undergo
through fork reversal (cruciform DNA intermediates) in tel1 cells, while in mre11 and sae2 cells, reversed forks
are further processed by nucleolytic events. In this study, we aim at investigating the mechanisms leading to
these pathological transitions at terminal forks. Fork reversal could be mediated by positive supercoiling
downstream of the forks. However, this is unlikely in our context as DSB formation should resolve the
topological constrains downstream of the fork. An alternative possibility is that fork reversal is mediated by
precatenane derivatives that intertwine the two replicated duplexes behind the replication fork. We tested this
possibility by overexpressing type II (TOP2) topoisomerase that should resolve precatenanes. Disrupting the
tethering of transcribed genes to the nuclear pore complex was shown to counteract fork reversal in
checkpoint-defective cells. We also tested whether nuclear envelope protein play any role in promoting
reversed forks formation. We will discuss the possibilities that may contribute to the mechanisms controlling
terminal fork integrity.
Mong Sing Lai 1 , Ylli Doksani 1 , Marco Foiani 1,2
1 FIRC Institute of Molecular Oncology Foundation (IFOM-IEO Campus), Via Adamello 16, 20139 Milan, Italy
2 DSBB-Universita degli Studi di Milano, Milan, Italy
14-17 June 2012, Heidelberg, Germany