Giuseppina D'Alessandro

First name
Giuseppina
Last name
D'Alessandro
Year of Study
Thesis Title
The role of RNA and DNA:RNA hybrids at DNA double-strand breaks
Thesis Abstract
The stability of our genome is constantly challenged by several genotoxic threats. DNA
double-strand breaks (DSBs) are the most dangerous DNA lesions that, if not repaired, can
lead to cancer initiation and progression and/or ageing. These detrimental consequences can
only be avoided if cells promptly recognize the lesions and signal their presence, thus
promoting either efficient repair and transient cell cycle arrest or cell death and cellular
senescence. This is the role of the DNA damage response (DDR) proteins and the newly
identified damage-induced non coding RNAs. We recently discovered that RNA polymerase
II is recruited to DSBs and synthetizes damage-induced non-coding RNAs (dilncRNAs).
DROSHA- and DICER-mediated processing of dilncRNAs generates small RNA species,
named DNA damage response RNA (DDRNAs) (Francia, 2012), that localize to DSBs via
pairing with dilncRNAs and promote DDR signaling (Michelini et al., in press). Similar
small non-coding RNA species discovered in plants are involved in DNA repair by
homologous recombination (HR) (Wei, 2012, Gao, 2014, Wang, 2016). In line with these
results, I report that transcriptional inhibition impairs recruitment of the HR proteins
BRCA1, BRCA2, and RAD51 to DSBs, while partially promoting DNA end resection.
Moreover, I show DNA:RNA hybrids accumulation at DSBs in mammalian cells by both
DRIP analyses and imaging techniques. Damage-induced DNA:RNA hybrids form upon the
hybridization of RNA species, likely dilncRNAs, to the resected DSBs DNA ends generated
during the S/G2 cell cycle phase. I also report that purified recombinant BRCA1 binds
DNA:RNA hybrids in vitro; moreover, DNA:RNA hybrids in vivo contribute to BRCA1
recruitment to DSBs. Consistent with the need to tightly regulate DNA:RNA hybrid levels,
I demonstrate that RNase H2, the major RNase H activity in mammalian nuclei, is recruited
to DSBs through direct interaction with RAD51. In summary, I report for the first time that
DNA:RNA hybrids accumulate at DSBs in mammalian cells in a cell-cycle- and DNA end
resection-depended way. At DSBs, BRCA1 directly recognizes DNA:RNA hybrids and
likely controls their turn-over by mediating the recruitment of RNase H2 via RAD51.
Students representatives
Off
Curricula Term