Proteomics

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PALB2 acetylation by KAT2A/B (GCN5/PCAF) in vitro

ABSTRACT: PArtner and Localizer of BRCA2 (PALB2) is essential to maintain genome stability in human cells. Upon DNA damage by double DNA strand breaks, PALB2 is required to repair DNA by homologous recombination. In undamaged conditions, PALB2 protects coding regions, by preventing DNA stress due to collisions between transcription and replication machineries. PALB2 associates with chromatin, which is essential to fulfill its function in genome stability maintenance, however molecular mechanisms regulating PALB2 chromatin association remain unknown. In our previous published study describing the KAT2A/B(GCN5/PCAF)-acetylome (Fournier et al., Nat.Comm. 2016, doi: 10.1038/ncomms13227) we have identified PALB2 as an acetylated protein target of the acetyltransferases KAT2A (GCN5) and KAT2B (PCAF) in vivo. KAT2A/B-acetylated sites of PALB2 were mapped within its DNA/Chromatin association domain. In this current study, we have conducted in vitro acetyltransferase (AT) assays, by mixing purified recombinant GST-tagged PALB2 full-length (FL) and PALB2 fragment P2.2 which associates with DNA/chromatin (from residues 295-610), with Flag-PCAF, Flag-GCN5 and Flag-GCN5 catalytic mutant (mut), followed by proteomics analysis to map PALB2 acetylated lysines by KAT2A(GCN5) and KAT2B(PCAF) in vitro. PALB2 FL and P2.2 alone, or PALB2 FL and P2.2 mixed with GCN5 catalytic mutant, were used as negative controls.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Marjorie Fournier  

LAB HEAD: Fumiko Esashi

PROVIDER: PXD014681 | Pride | 2023-03-10

REPOSITORIES: Pride

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Dataset's files

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Action DRS
PALB2 P2.2+GCN5_1.msf Msf
PALB2 P2.2+GCN5_1.raw Raw
PALB2-FLGCN5_1.msf Msf
PALB2-FLGCN5_1.raw Raw
PALB2-FLGCN5mut_1.msf Msf
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Publications

The tumour suppressor PALB2 stimulates RAD51-mediated homologous recombination (HR) repair of DNA damage, whilst its steady-state association with active genes protects these loci from replication stress. Here, we report that the lysine acetyltransferases 2A and 2B (KAT2A/2B, also called GCN5/PCAF), two well-known transcriptional regulators, acetylate a cluster of seven lysine residues (7K-patch) within the PALB2 chromatin association motif (ChAM) and, in this way, regulate context-dependent PAL  ...[more]

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