Kinome-wide Screening Uncovers A Role for Bromodomain Protein 3 in DNA Double-stranded Break Repair

Most living organisms have evolved at least two discrete mechanisms to repair DNA double-stranded breaks (DSBs): homology dependent recombination (HDR) and classical non-homologous end joining (C-NHEJ). In HDR, a DSB is repaired using an undamaged homologue or sister chromatid in a process that generally requires extensive regions of homology between the damaged chromosome and the undamaged donor [1], [2]. Most mitotic and meiotic recombination and the repair of DSBs in late S and G2 phases of the cell cycle [e.g., stalled replication forks; [3]] are carried out by HDR. Thus, HDR accounts for an important portion of the DNA DSB activity in a wild-type human cell. Not surprisingly therefore, mutations of many HDR genes are associated with cancer predisposition in humans [4], [5]. One of the most critical HDR genes is Radiation Sensitive 51 (RAD51), which is responsible for the homology searches and strand exchanges required during HDR [6].

The bulk of DSB repair in higher eukaryotes proceeds, however, more frequently by a process that does not require large regions of homology. Specifically, mammalian cells have evolved an efficient ability to join nonhomologous DNA molecules together [7] using the C-NHEJ pathway [8]. C-NHEJ is critically required for the proper development of the immune system and it is especially clinically relevant because it is the preferred pathway that cells utilize to repair ionizing radiation (IR)-induced DSBs [9], [10]. Three important C-NHEJ genes are the protein kinase, DNA-activated, catalytic subunit (PRKDC or DNA-PKcs), and its DNA binding heterodimeric subunit, Ku70:Ku86 [11], [12]. Relevantly, mutations in PRKDC have been associated with IRs (IR sensitivity), immune deficiency and/or cancer predisposition in humans [13], [14], [15].

Although much work has been carried out and great progress has been made over the last decade [16], [17], a complete mechanistic understanding of how a cell decides to repair a DSB via HDR or C-NHEJ is still lacking. The mammalian DNA damage response (DDR) is regulated by PRKDC and the related kinases ataxia telangiectasia mutated (ATM), and ataxia telangiectasia and RAD3 related (ATR) [18], [19]. Some of the relevant substrates for these kinases have been identified, but their pathways are also still not completely defined. In the past, this problem has been addressed by knocking out or knocking down the expression of ATM, ATR, and/or PRKDC in a favorite model system and then identifying those proteins phosphorylated in the control, but not the treated, cells following IR exposure [20]. Alternatively, mutations were made in one of these kinases and then mRNA was quantitated to identify genes that were either up or down regulated plus or minus IR exposure [21], [22], [23]. We have taken a unique approach to this problem. First, we generated human cell models for PRKDC including a knockout [24] and a kinase-dead knock-in [25]. We then used the kinase-dead and the parental cell line to perform "kinome" analyses [26] following IR exposure. In this approach, cellular extracts are incubated with beads onto which a dozen ATP-analog inhibitors have been immobilized. Since every kinase has an ATP-binding domain, these beads bind mostly kinases and other proteins containing ATP-binding domains (e.g., ATPases, chromatin remodelers, etc.). The beads are then centrifuged down, washed and subjected to quantitative proteomics. In this fashion, ~70% of all human kinases and a multitude of additional ATP-binding proteins can be queried [26]. These analyses identified BRD3 (bromodomain containing protein 3) as a protein whose expression was almost completely downregulated in the PRKDC kinase-dead cell line. Importantly, although BRD3 is not a kinase, it is likely an ATP-dependent chromatin remodeler [27] and we believe that either its putative ATP-interaction domain [28] or its interaction with a bona fide ATP-binding protein, allowed it to be identified by this screen.

There are 46 bromodomain-containing proteins (BRDs) encoded in the human genome. A BRD subfamily, called the bromodomain (BD) and extra terminal (ET) domain (BET) family, consists of 4 members [BRD2, BRD3, BRD4, and BRDT (T = testis specific)] each of which contains two N-terminal BD domains and an “extra” C-terminal domain [29], [30]. BDs are modules that facilitate a protein’s ability to bind to acetylated lysines [31]. Since acetylation is often associated with histone modification it is not surprising that the majority of BRDs (including the BET family members) are involved in chromatin regulation and transcription [27], [32]. The most intensely researched BET family member is BRD4, an important transcription factor [33] and a protein that has been implicated in the DDR [34], [35] potentially by preventing the accumulation of R-loops [36]. Most recently, BRD4 has also been directly implicated in HDR-mediated DSB repair using an in vitro Xenopus extract system [37].

In stark contrast, there is little known about BRD3 and there are no reports of BRD3’s involvement in HDR. To date, the best function for BRD3 was inferred from an isolation of proteins on nascent DNA analysis where it was determined that BRD3 can bind to and regulate a protein called ATPase Family AAA domain containing 5 [ATAD5; [38]]. ATAD5, in turn, is the main component of a complex that unloads proliferating nuclear cell antigen (PCNA) from replicating DNA. Thus, BRD3, through its interaction with ATAD5, negatively regulates the amount of PCNA on a replication fork via ATAD5’s PCNA unloading activity [38], [39], [40], [41]. Other than these few reports, however, there is little biochemical information available about BRD3.

Here we demonstrate that BRD3-null human cells are hyper-recombinogenic. Thus, human cells that have been genetically engineered to lack BRD3 expression show elevated levels of gene conversion, sister chromatid exchanges and gene targeting. Biochemically, this increase in HDR activity appears to be related to the altered kinetics of RAD51 recruitment at DSBs. Thus, we have identified BRD3 as a potent negative regulator of human HDR and this function may explain why BRD3 expression was specifically ablated in PRKDC/DNA-PKcs kinase-dead cells, which are incapable of performing C-NHEJ.

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