The activity of human replication protein A (RPA) in DNA replication and repair is regulated by phosphorylation of the middle RPA2 subunit. It has previously been shown that up to nine different N-terminal residues are modified in vivo and in response to genotoxic stress. Using a novel antibody against phospho-Ser 29 , a moiety formed by cyclin-Cdk, we observed that RPA2 was phosphorylated during mitosis in nonstressed cells. Robust phosphorylation of Ser 29 was also seen in interphase cells following treatment with the DNA-damaging agent camptothecin, a rare example of stress stimulating the modification of a repair factor by cyclin-Cdk. RPA2 phosphorylation is regulated both in cis and trans. Cis-phosphorylation follows a preferred pathway. (That is, the initial modification of Ser 33 by ATR stimulates subsequent phosphorylation of Cdk sites Ser 23 and Ser 29 ). These events then facilitate modification of Thr 21 and extreme N-terminal sites Ser 4 and Ser 8 , probably by DNA-PK. Our data also indicate that the phosphorylation of one RPA molecule can influence the phosphorylation of other RPA molecules in trans. Cells in which endogenous RPA2 was "replaced" with a double S23A/S29A-RPA2 mutant were seen to have an abnormal cell cycle distribution both in normal and in stressed cells. Such cells also showed aberrant DNA damage-dependent RPA foci and had persistent staining of ␥H2AX following DNA damage. Our data indicate that RPA phosphorylation facilitates chromosomal DNA repair. We postulate that the RPA phosphorylation pattern provides a means to regulate the DNA repair pathway utilized.Replication protein A (RPA) 2 is a heterotrimeric singlestranded DNA-binding factor that is critical for the "three Rs" of eukaryotic DNA enzymology: DNA replication, DNA recombination, and DNA repair (1, 2). For DNA replication, the study of cellular and viral model systems demonstrates that RPA is needed both for origin denaturation and replication elongation, in the latter case to facilitate the switch from DNA polymerase ␣ to DNA polymerase ␦ during Okazaki fragment synthesis (3). RPA acts in homologous recombination (HR) to stimulate DNA annealing using physical interactions with Rad52 (4 -7) and in HR-mediated DNA repair, probably employing specific interactions with BRCA2 (8, 9). RPA is a required factor in both the nucleotide excision (10, 11) and mismatch repair pathways (12, 13) and in somatic hypermutation (14). Because of these many roles, it is of significant interest to understand the mechanisms that regulate RPA activity.Of the ϳ70-kDa (RPA1), 30-kDa (RPA2), and 14-kDa (RPA3) subunits, human RPA is subject to extensive phosphorylation on RPA2 (2) and at one RPA1 site (15). The N-terminal 33 residues of RPA2 undergo both cell cycle-and stress-dependent phosphorylation on approximately nine sites (Fig. 1A), which are thought to exist in an unstructured conformation (16,17). Ser 23 and Ser 29 are constitutively modified during mitosis by cyclin B-Cdk1 (18, 19) and have been suggested to be partially modified beginni...

under non-stress conditions, the mutant cells were severely deficient in the amount of DNA synthesis occurring during replication stress. These cells also had abnormally high levels of chromatin-bound RPA, indicative of increased amounts of single-stranded DNA (ssDNA) and showed defective recovery from stress. Cells replaced with the mutant RPA2 also generated G1 cells with a broader DNA distribution and high levels of apoptosis following stress, compared with cells expressing wild-type RPA2. Surprisingly, cells expressing the wild-type RPA2 subunit had increased levels of stress-dependent DNA breaks. Our data demonstrate that RPA phosphorylation at the T21 and S33 sites facilitates adaptation of a DNA-replication fork to replication stress. Supplementary material available online at

BRCA1 plays a critical role in homology-directed repair (HDR) of DNA double strand breaks, and the repair defect of BRCA1-mutant cancer cells is being targeted with platinum drugs and poly (ADP-ribose) polymerase (PARP) inhibitors. We have employed relatively simple and sensitive assays to determine the function of BRCA1 variants or mutants in two HDR mechanisms, homologous recombination (HR) and single strand annealing (SSA), and in conferring resistance to cisplatin and olaparib in human cancer cells. Our results define the functionality of the top 22 patient-derived BRCA1 missense variants and the contribution of different domains of BRCA1 and its E3 ubiquitin ligase activity to HDR and drug resistance. Importantly, our results also demonstrate that the BRCA1-PALB2 interaction dictates the choice between HR and SSA. These studies establish functional and mutational landscapes of BRCA1 for HDR and therapy resistance, while revealing novel insights into BRCA1 regulatory mechanisms and HDR pathway choice.DOI: http://dx.doi.org/10.7554/eLife.21350.001

Human replication protein A (RPA) becomes phosphorylated on the RPA2 subunit by cyclin B-Cdc2 during mitosis, although the functional role of this modification is unclear. We find that this modification stimulates RPA2 to become hyperphosphorylated in response to mitotic DNA damage caused by bleomycin treatment. Cells in which endogenous RPA2 was replaced by a mutant subunit lacking both Cdc2 sites had a significant defect in mitotic release into a 2N G1 phase after exposure to bleomycin. An increased percentage of these mutant cells also was positive initially for cyclin B expression and BubR1 chromatin staining, indicative of an extended spindle assembly checkpoint. The mutant cells that experienced mitotic DNA damage also underwent apoptosis at higher levels than cells expressing the WT subunit. Even so, we did not find the mutation had any dramatic effects on the level of DNA repair in mitosis. Cells lacking ATM (a checkpoint factor and RPA2 kinase) also were severely defective in mitotic exit and were unable to support RPA hyperphosphorylation after mitotic DNA damage. Although checkpoint 1 effector kinase (Chk1) had a more complex role, inhibition of Chk1 activity with UCN-01 also reduced mitotic exit. Chk1 activation and mitotic RPA hyperphosphorylation were found to be independent events. Our results demonstrate that mitotic RPA hyperphosphorylation facilitates release of cells from a damaged mitosis into a 2N G1 phase, thereby increasing cell viability. DNA damage occurring during mitosis represents a daunting challenge to the integrity of the mammalian genome. Condensation of chromosomal DNA in preparation for cytokinesis constrains the activity of DNA repair reactions. Nevertheless, double-strand DNA breaks (DSBs) induced by exposure of prometaphase cells to ionizing radiation or topoisomerase II inhibitors cause a delay in mitotic exit (1, 2). The nature of this delay and its causes are only poorly understood.An essential DNA replication factor that is involved intimately in the signaling and repair of DNA damage is replication protein A (RPA), a heterotrimeric single-stranded DNA-binding complex with subunits in human cells of 70 kDa (RPA1), 30 kDa (RPA2), and 14 kDa (RPA3) (3, 4). RPA acts during DNA replication stress conditions to recruit and activate the checkpoint kinase ATR (5, 6). The N terminus of the human RPA2 subunit also is a target of ATR and other members of the phosphatidylinositol 3-kinase-like kinase family (PIKKs; e.g., ATM and DNA-PK) and cyclin-Cdk complexes ( Fig. 1A; e.g., refs. 7-14). RPA2 phosphorylation follows a preferred pathway so that initial modification by a kinase (e.g., cyclin A-Cdk2) stimulates the subsequent modification by another kinase (e.g., DNA-PK) (14). Recent cell-based studies have shown that human RPA activity in DNA replication and repair is regulated by changes in the pattern of RPA phosphorylation. For example, human cells in which endogenous RPA2 has been replaced with a RPA2 variant mutated at the two RPA2 cyclin-Cdk sites (S23A/ S29A-RPA2) are defec...

BackgroundHeterogeneous nuclear ribonucleoprotein C1/C2 (hnRNP C) is a core component of 40S ribonucleoprotein particles that bind pre-mRNAs and influence their processing, stability and export. Breast cancer tumor suppressors BRCA1, BRCA2 and PALB2 form a complex and play key roles in homologous recombination (HR), DNA double strand break (DSB) repair and cell cycle regulation following DNA damage.MethodsPALB2 nucleoprotein complexes were isolated using tandem affinity purification from nuclease-solubilized nuclear fraction. Immunofluorescence was used for localization studies of proteins. siRNA-mediated gene silencing and flow cytometry were used for studying DNA repair efficiency and cell cycle distribution/checkpoints. The effect of hnRNP C on mRNA abundance was assayed using quantitative reverse transcriptase PCR.Results and SignificanceWe identified hnRNP C as a component of a nucleoprotein complex containing breast cancer suppressor proteins PALB2, BRCA2 and BRCA1. Notably, other components of the 40S ribonucleoprotein particle were not present in the complex. hnRNP C was found to undergo significant changes of sub-nuclear localization after ionizing radiation (IR) and to partially localize to DNA damage sites. Depletion of hnRNP C substantially altered the normal balance of repair mechanisms following DSB induction, reducing HR usage in particular, and impaired S phase progression after IR. Moreover, loss of hnRNP C strongly reduced the abundance of key HR proteins BRCA1, BRCA2, RAD51 and BRIP1, which can be attributed, at least in part, to the downregulation of their mRNAs due to aberrant splicing. Our results establish hnRNP C as a key regulator of BRCA gene expression and HR-based DNA repair. They also suggest the existence of an RNA regulatory program at sites of DNA damage, which involves a unique function of hnRNP C that is independent of the 40S ribonucleoprotein particles and most other hnRNP proteins.