How do cells sense DNA lesions?

Colombo, Chiara Vittoria; Gnugnoli, Marco; Gobbini, Elisa; Longhese, Maria Pia

doi:10.1042/bst20191118

Cited by 16 publications

(13 citation statements)

References 164 publications

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“…The preservation of genome integrity is vital for the proper development of an organism. Cells are subjected to multiple endogenous and exogenous agents capable of causing lesions and affect multiple DNA transaction processes (e.g., replication, repair, and transcription) (1). Covalent DNA-protein cross-links are bulky lesions and can be toxic if left unrepaired (2); they are formed from both exogenous and endogenous sources.…”

mentioning

confidence: 99%

Enzymatic bypass of an N6-deoxyadenosine DNA–ethylene dibromide–peptide cross-link by translesion DNA polymerases

Ghodke¹,

Gonzalez-Vasquez²,

Wang

et al. 2021

Journal of Biological Chemistry

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Unrepaired DNA–protein cross-links, due to their bulky nature, can stall replication forks and result in genome instability. Large DNA–protein cross-links can be cleaved into DNA–peptide cross-links, but the extent to which these smaller fragments disrupt normal replication is not clear. Ethylene dibromide (1,2-dibromoethane) is a known carcinogen that can cross-link the repair protein O 6 -alkylguanine-DNA alkyltransferase (AGT) to the N6 position of deoxyadenosine (dA) in DNA, as well as four other positions in DNA. We investigated the effect of a 15-mer peptide from the active site of AGT, cross-linked to the N6 position of dA, on DNA replication by human translesion synthesis DNA polymerases (Pols) η, ⍳, and κ. The peptide–DNA cross-link was bypassed by the three polymerases at different rates. In steady-state kinetics, the specificity constant ( k cat / K m ) for incorporation of the correct nucleotide opposite to the adduct decreased by 220-fold with Pol κ, tenfold with pol η, and not at all with Pol ⍳. Pol η incorporated all four nucleotides across from the lesion, with the preference dT > dC > dA > dG, while Pol ⍳ and κ only incorporated the correct nucleotide. However, LC-MS/MS analysis of the primer-template extension product revealed error-free bypass of the cross-linked 15-mer peptide by Pol η. We conclude that a bulky 15-mer peptide cross-linked to the N6 position of dA can retard polymerization and cause miscoding but that overall fidelity is not compromised because only correct pairs are extended.

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confidence: 99%

Enzymatic bypass of an N6-deoxyadenosine DNA–ethylene dibromide–peptide cross-link by translesion DNA polymerases

Ghodke¹,

Gonzalez-Vasquez²,

Wang

et al. 2021

Journal of Biological Chemistry

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“…We next tested the respective contribution of the two H2A kinases Mec1 and Tel1 in this phosphorylation event since Tel1 is the main kinase responsible for H2A-P at telomeres ([ 45 ] and Figure S5C ), whereas Mec1 is the main kinase acting at processed DSB [ 47 ]. While deleting TEL1 had no significant effect on the kinetic and level of H2A-P, deleting MEC1 led to a reduction in H2A-P ( Figure 5 B) upon LacI induction compared to the wild-type strain.…”

Section: Resultsmentioning

confidence: 99%

An Inducible System for Silencing Establishment Reveals a Stepwise Mechanism in Which Anchoring at the Nuclear Periphery Precedes Heterochromatin Formation

Loïodice

Garnier

Nikolov

et al. 2021

Cells

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In eukaryotic cells, silent chromatin is mainly found at the nuclear periphery forming subnuclear compartments that favor silencing establishment. Here, we set up an inducible system to monitor silencing establishment at an ectopic locus in relation with its subnuclear localization in budding yeast. We previously showed that introducing LacI bound lacO arrays in proximity to gene flanked by HML silencers favors the recruitment of the yeast silencing complex SIR at this locus, leading to its silencing and anchoring at the nuclear periphery. Using an inducible version of this system, we show that silencing establishment is a stepwise process occurring over several cell cycles, with the progressive recruitment of the SIR complex. In contrast, we observed a rapid, SIR-independent perinuclear anchoring, induced by the high amount of LacI binding at the lacO array leading to nucleosome eviction at this array and to the phosphorylation of H2A in the neighboring nucleosomes by Mec1 kinase. While the initial phosphorylation of H2A (H2A-P) and perinuclear anchoring are independent of the SIR complex, its latter recruitment stabilizes H2A-P and reinforces the perinuclear anchoring. Finally, we showed that Sir3 spreading stabilizes nucleosomes and limits the access of specific DNA-binding protein to DNA.

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“…ATM and ATR are two checkpoint kinases which regulate the cellular response to genotoxic stress [ 18 – 22 ]. ATM has been traditionally studied in the context of double-strand breaks, and TIP60 was shown to be specifically required for ATM activation, by catalyzing an activatory acetylation of ATM at Lys3016 [ 23 , 24 ].…”

Section: Introductionmentioning

confidence: 99%

The TIP60-ATM axis regulates replication fork stability in BRCA-deficient cells

et al. 2022

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Maintenance of replication fork stability is essential for genome preservation. Stalled replication forks can be reversed by translocases such as SMARCAL1, and unless protected through the activity of the BRCA pathway, are subsequently subjected to nucleolytic degradation. The ATM and ATR kinases are master regulators of the DNA damage response. ATM activation upon DNA damage is mediated by the acetyltransferase TIP60. Here, we show that the TIP60-ATM pathway promotes replication fork reversal by recruiting SMARCAL1 to stalled forks. This enables fork degradation in BRCA-deficient cells. We also show that this ATM activity is not shared by ATR. Moreover, we performed a series of genome-wide CRISPR knockout genetic screens to identify genetic determinants of the cellular sensitivity to ATM inhibition in wildtype and BRCA2-knockout cells, and validated the top hits from multiple screens. We provide a valuable list of common genes which regulate the response to multiple ATM inhibitors. Importantly, we identify a differential response of wildtype and BRCA2-deficient cells to these inhibitors. In BRCA2-knockout cells, DNA repair genes (including RAD17, MDC1, and USP28) were essential for survival upon ATM inhibitor treatment, which was not the case in wild-type cells. These findings may eventually help guide the way for rational deployment of ATM inhibitors in the clinic.

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How do cells sense DNA lesions?

Cited by 16 publications

References 164 publications

Enzymatic bypass of an N6-deoxyadenosine DNA–ethylene dibromide–peptide cross-link by translesion DNA polymerases

Enzymatic bypass of an N6-deoxyadenosine DNA–ethylene dibromide–peptide cross-link by translesion DNA polymerases

An Inducible System for Silencing Establishment Reveals a Stepwise Mechanism in Which Anchoring at the Nuclear Periphery Precedes Heterochromatin Formation

The TIP60-ATM axis regulates replication fork stability in BRCA-deficient cells

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