Many proteins that respond to DNA damage are recruited to DNA lesions. We used a proteomics approach that coupled isotopic labeling with chromatin fractionation and mass spectrometry to uncover proteins that associate with damaged DNA, many of which are involved in DNA repair or nucleolar function. We show that polycomb group members are recruited by poly(ADP ribose) polymerase (PARP) to DNA lesions following UV laser microirradiation. Loss of polycomb components results in IR sensitivity of mammalian cells and Caenorhabditis elegans. PARP also recruits two components of the repressive nucleosome remodeling and deacetylase (NuRD) complex, chromodomain helicase DNA-binding protein 4 (CHD4) and metastasis associated 1 (MTA1), to DNA lesions. PARP plays a role in removing nascent RNA and elongating RNA polymerase II from sites of DNA damage. We propose that PARP sets up a transient repressive chromatin structure at sites of DNA damage to block transcription and facilitate DNA repair. T he cellular response to DNA damage is initiated by the sensing of structural alterations in DNA that culminates in the activation of phosphoinositide-3-kinase-related protein kinases (PIKKs) that include the ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) kinases (1). With the help of mediators, ATM and ATR subsequently signal downstream to activate effector kinases checkpoint 1 (CHK1) and checkpoint 2 (CHK2), leading to transcriptional induction, cell-cycle arrest, DNA repair, senescence, or apoptosis. This DNA damage response induces the sequential recruitment of an extensive network of proteins to the sites of damage. For example, in response to double-strand breaks (DSBs), ATM phosphorylates histone H2AX adjacent to the break to initiate a H2AX-dependent concentration of proteins involved in the DNA damage response, such as mediator of DNA damage checkpoint protein 1 (MDC1), which recruits additional molecules of the ATM kinase. This recruitment effectively initiates a positive feedback loop that promotes the spread of γH2AX-flanking DSBs (2). Phosphorylation of MDC1 by ATM creates a motif that is recognized by the ubiquitin ligase ring finger 8 (RNF8) (3-6) that, with the help of ring finger 168 (RNF168), catalyzes the formation of lysine 63 (K63)-linked polyubiquitin chains that ultimately recruit the breast cancer 1 (BRCA1) A complex containing receptor-associated protein 80 (RAP80), Abraxas, BRCA1, new component of the BRCA1 A complex (NBA1), and BRCA1/BRCA2-containing complex, subunit 3 (BRCC36) (3-10) as well as p53 binding protein 1 (53BP1) and RAD18 homolog (RAD18) (3-8, 11).Several factors, such as Nijmegen breakage syndrome 1 (NBS1), 53BP1, and BRCA1, are recruited to the sites of damage in an H2AX-independent manner (12). However, these interactions appear to be more transient and may play a role as an initial response to DNA damage that is distinct from the extended association of factors via γH2AX. Several additional pathways also have been shown to direct the recruitment of vario...

The abundance of cellular proteins is determined largely by the rate of transcription and translation coupled with the stability of individual proteins. Although we know a great deal about global transcript abundance, little is known about global protein stability. We present a highly parallel multiplexing strategy to monitor protein turnover on a global scale by coupling flow cytometry with microarray technology to track the stability of individual proteins within a complex mixture. We demonstrated the feasibility of this approach by measuring the stability of approximately 8000 human proteins and identifying proteasome substrates. The technology provides a general platform for proteome-scale analysis of protein turnover under various physiological and disease conditions.