Featured Publication

Rajashree A Deshpande, Logan R Myler, Michael M Soniat, Nodar Makharashvili, Linda Lee, Susan P Lees-Miller, Ilya J Finkelstein, and Tanya T Paull. 2AD. “DNA-dependent protein kinase promotes DNA end processing by MRN and CtIP.” Sci Adv, 6, 2, Pp. eaay0922. Abstract
The repair of DNA double-strand breaks occurs through nonhomologous end joining or homologous recombination in vertebrate cells-a choice that is thought to be decided by a competition between DNA-dependent protein kinase (DNA-PK) and the Mre11/Rad50/Nbs1 (MRN) complex but is not well understood. Using ensemble biochemistry and single-molecule approaches, here, we show that the MRN complex is dependent on DNA-PK and phosphorylated CtIP to perform efficient processing and resection of DNA ends in physiological conditions, thus eliminating the competition model. Endonucleolytic removal of DNA-PK-bound DNA ends is also observed at double-strand break sites in human cells. The involvement of DNA-PK in MRN-mediated end processing promotes an efficient and sequential transition from nonhomologous end joining to homologous recombination by facilitating DNA-PK removal.
Logan R Myler, Michael M Soniat, Xiaoming Zhang, Rajashree A Deshpande, Tanya T Paull, and Ilya J Finkelstein. 2AD. “Purification and Biophysical Characterization of the Mre11-Rad50-Nbs1 Complex.” Methods Mol Biol, 2004, Pp. 269-287. Abstract
The Mre11-Rad50-Nbs1 (MRN) complex coordinates the repair of DNA double-strand breaks, replication fork restart, meiosis, class-switch recombination, and telomere maintenance. As such, MRN is an essential molecular machine that has homologs in all organisms of life, from bacteriophage to humans. In human cells, MRN is a >500 kDa multifunctional complex that encodes DNA binding, ATPase, and both endonuclease and exonuclease activities. MRN also forms larger assemblies and interacts with multiple DNA repair and replication factors. The enzymatic properties of MRN have been the subject of intense research for over 20 years, and more recently, single-molecule biophysics studies are beginning to probe its many biochemical activities. Here, we describe the methods used to overexpress, fluorescently label, and visualize MRN and its activities on single molecules of DNA.
Chung-Hsuan Kao, Seung W Ryu, Min J Kim, Xuemei Wen, Oshadi Wimalarathne, and Tanya T Paull. 2AD. “Growth-Regulated Hsp70 Phosphorylation Regulates Stress Responses and Prion Maintenance.” Mol Cell Biol, 40, 12. Abstract
Maintenance of protein homeostasis in eukaryotes under normal growth and stress conditions requires the functions of Hsp70 chaperones and associated cochaperones. Here, we investigate an evolutionarily conserved serine phosphorylation that occurs at the site of communication between the nucleotide-binding and substrate-binding domains of Hsp70. Ser151 phosphorylation in yeast Hsp70 (Ssa1) is promoted by cyclin-dependent kinase (Cdk1) during normal growth. Phosphomimetic substitutions at this site (S151D) dramatically downregulate heat shock responses, a result conserved with HSC70 S153 in human cells. Phosphomimetic forms of Ssa1 also fail to relocalize in response to starvation conditions, do not associate with Hsp40 cochaperones Ydj1 and Sis1, and do not catalyze refolding of denatured proteins in cooperation with Ydj1 and Hsp104. Despite these negative effects on HSC70/HSP70 function, the S151D phosphomimetic allele promotes survival of heavy metal exposure and suppresses the Sup35-dependent [ ] prion phenotype, consistent with proposed roles for Ssa1 and Hsp104 in generating self-nucleating seeds of misfolded proteins. Taken together, these results suggest that Cdk1 can downregulate Hsp70 function through phosphorylation of this site, with potential costs to overall chaperone efficiency but also advantages with respect to reduction of metal-induced and prion-dependent protein aggregate production.
Michael M Soniat, Logan R Myler, Hung-Che Kuo, Tanya T Paull, and Ilya J Finkelstein. 2AD. “RPA Phosphorylation Inhibits DNA Resection.” Mol Cell, 75, 1, Pp. 145-153.e5. Abstract
Genetic recombination in all kingdoms of life initiates when helicases and nucleases process (resect) the free DNA ends to expose single-stranded DNA (ssDNA) overhangs. Resection regulation in bacteria is programmed by a DNA sequence, but a general mechanism limiting resection in eukaryotes has remained elusive. Using single-molecule imaging of reconstituted human DNA repair factors, we identify phosphorylated RPA (pRPA) as a negative resection regulator. Bloom's syndrome (BLM) helicase together with exonuclease 1 (EXO1) and DNA2 nucleases catalyze kilobase-length DNA resection on nucleosome-coated DNA. The resulting ssDNA is rapidly bound by RPA, which further stimulates DNA resection. RPA is phosphorylated during resection as part of the DNA damage response (DDR). Remarkably, pRPA inhibits DNA resection in cellular assays and in vitro via inhibition of BLM helicase. pRPA suppresses BLM initiation at DNA ends and promotes the intrinsic helicase strand-switching activity. These findings establish that pRPA provides a feedback loop between DNA resection and the DDR.
Seung W Ryu, Rose Stewart, Chase D Pectol, Nicolette A Ender, Oshadi Wimalarathne, Ji-hoon Lee, Carlos P Zanini, Antony Harvey, Jon M Huibregtse, Peter Mueller, and Tanya T Paull. 2AD. “Proteome-wide identification of HSP70/HSC70 chaperone clients in human cells.” PLoS Biol, 18, 7, Pp. e3000606. Abstract
The 70 kDa heat shock protein (HSP70) family of chaperones are the front line of protection from stress-induced misfolding and aggregation of polypeptides in most organisms and are responsible for promoting the stability, folding, and degradation of clients to maintain cellular protein homeostasis. Here, we demonstrate quantitative identification of HSP70 and 71 kDa heat shock cognate (HSC70) clients using a ubiquitin-mediated proximity tagging strategy and show that, despite their high degree of similarity, these enzymes have largely nonoverlapping specificities. Both proteins show a preference for association with newly synthesized polypeptides, but each responds differently to changes in the stoichiometry of proteins in obligate multi-subunit complexes. In addition, expression of an amyotrophic lateral sclerosis (ALS)-associated superoxide dismutase 1 (SOD1) mutant protein induces changes in HSP70 and HSC70 client association and aggregation toward polypeptides with predicted disorder, indicating that there are global effects from a single misfolded protein that extend to many clients within chaperone networks. Together these findings show that the ubiquitin-activated interaction trap (UBAIT) fusion system can efficiently isolate the complex interactome of HSP chaperone family proteins under normal and stress conditions.
Ji-hoon Lee, Seung W Ryu, Nicolette A Ender, and Tanya T Paull. 2AD. “Poly-ADP-ribosylation drives loss of protein homeostasis in ATM and Mre11 deficiency.” Mol Cell, 81, 7, Pp. 1515-1533.e5. Abstract
Loss of the ataxia-telangiectasia mutated (ATM) kinase causes cerebellum-specific neurodegeneration in humans. We previously demonstrated that deficiency in ATM activation via oxidative stress generates insoluble protein aggregates in human cells, reminiscent of protein dysfunction in common neurodegenerative disorders. Here, we show that this process is driven by poly-ADP-ribose polymerases (PARPs) and that the insoluble protein species arise from intrinsically disordered proteins associating with PAR-associated genomic sites in ATM-deficient cells. The lesions implicated in this process are single-strand DNA breaks dependent on reactive oxygen species, transcription, and R-loops. Human cells expressing Mre11 A-T-like disorder mutants also show PARP-dependent aggregation identical to ATM deficiency. Lastly, analysis of A-T patient cerebellum samples shows widespread protein aggregation as well as loss of proteins known to be critical in human spinocerebellar ataxias that is not observed in neocortex tissues. These results provide a hypothesis accounting for loss of protein integrity and cerebellum function in A-T.