By coupling the protection and organization of single-stranded DNA (ssDNA) with recruitment and alignment of DNA processing factors, replication protein A (RPA) lies at the heart of dynamic multi-protein DNA processing machinery. Nevertheless, how RPA coordinates biochemical functions of its eight domains remains unknown. We examined the structural biochemistry of RPA’s DNA-binding activity, combining small-angle X-ray and neutron scattering with all-atom molecular dynamics simulations to investigate the architecture of RPA’s DNA-binding core. The scattering data reveal compaction promoted by DNA binding; DNA-free RPA exists in an ensemble of states with inter-domain mobility and becomes progressively more condensed and less dynamic on binding ssDNA. Our results contrast with previous models proposing RPA initially binds ssDNA in a condensed state and becomes more extended as it fully engages the substrate. Moreover, the consensus view that RPA engages ssDNA in initial, intermediate and final stages conflicts with our data revealing that RPA undergoes two (not three) transitions as it binds ssDNA with no evidence for a discrete intermediate state. These results form a framework for understanding how RPA integrates the ssDNA substrate into DNA processing machinery, provides substrate access to its binding partners and promotes the progression and selection of DNA processing pathways.

Small-angle X-ray scattering (SAXS) has emerged as an enabling integrative technique for comprehensive analyses of macromolecular structures and interactions in solution. Over the past two decades, SAXS has become a mainstay of the structural biologist’s toolbox, supplying multiplexed measurements of molecular shape and dynamics that unveil biological function. Here, we discuss evolving SAXS theory, methods, and applications that extend the field of small-angle scattering beyond simple shape characterization. SAXS, coupled with size-exclusion chromatography (SEC-SAXS) and time-resolved (TR-SAXS) methods, is now providing high-resolution insight into macromolecular flexibility and ensembles, delineating biophysical landscapes, and facilitating high-throughput library screening to assess macromolecular properties and to create opportunities for drug discovery. Looking forward, we consider SAXS in the integrative era of hybrid structural biology methods, its potential for illuminating cellular supramolecular and mesoscale structures, and its capacity to complement high-throughput bioinformatics sequencing data. As advances in the field continue, we look forward to proliferating uses of SAXS based upon its abilities to robustly produce mechanistic insights for biology and medicine.

In response to DNA damage, eukaryotic cells activate a series of DNA damage-dependent pathways that serve to arrest cell cycle progression and remove DNA damage. Coordination of cell cycle arrest and damage repair is critical for maintenance of genomic stability. However, this process is still poorly understood. Nucleotide excision repair (NER) and the ATR-dependent cell cycle checkpoint are the major pathways responsible for repair of UV-induced DNA damage. Here we show that ATR physically interacts with the NER factor Xeroderma pigmentosum group A (XPA). Using a mass spectrometry-based protein footprinting method, we found that ATR interacts with a helixturn-helix motif in the minimal DNA-binding domain of XPA where an ATR phosphorylation site (serine 196) is located. XPAdeficient cells complemented with XPA containing a point mutation of S196A displayed a reduced repair efficiency of cyclobutane pyrimidine dimers as compared with cells complemented with wild-type XPA, although no effect was observed for repair of (6-4) photoproducts. This suggests that the ATR-dependent phosphorylation of XPA may promote NER repair of persistent DNA damage. In addition, a K188A point mutation of XPA that disrupts the ATR-XPA interaction inhibits the nuclear import of XPA after UV irradiation and, thus, significantly reduced DNA repair efficiency. By contrast, the S196A mutation has no effect on XPA nuclear translocation. Taken together, our results suggest that the ATR-XPA interaction mediated by the helix-turn-helix motif of XPA plays an important role in DNA-damage responses to promote cell survival and genomic stability after UV irradiation.

Modular proteins with multiple domains tethered by flexible linkers have variable global archiectures. Using the eukaryotic ssDNA binding protein, Replication Protein A (RPA), we demonstrate that NMR spectroscopy is a powerful tool to characterize the remodeling of architecture in different functional states. The first direct evidence is obtained for the remodeling of RPA upon binding ssDNA, including an alteration in the availability of the RPA32N domain that may help explain its damage-dependent phosphorylation.The progression of DNA replication and repair requires the coordinated action of dynamic, multi-protein assemblies. We have previously proposed a critical role for proteins composed of multiple, flexibly attached domains in facilitating the action of these dynamic complexes 1 . Because these proteins can undergo intra-and inter-domain rearrangements, they are able to interact optimally with the ever-changing substrate landscape present during DNA processing. RPA is a prototypical modular multi-domain DNA processing protein with flexible linkers of various lengths (Figure 1). The trimer core is a compact assembly of three OB-fold domains (RPA70C/32D/14) to which is appended the disordered RPA32N functional domain, the RPA32C winged-helix domain, and the tandem RPA70AB and the RPA70N OB-fold NMR spectroscopy in solution is a powerful tool for characterizing proteins under conditions that preserve intrinsic dynamic properties. The advent of TROSY, CRINEPT and related experimental approaches 3 has vastly increased the upper limit of molecular masses accessible to study by NMR. Examples range from the globular malate synthase (82 kDa) to the oligomeric GroEL-GroES complex (872 kDa) to highly flexible domains from the ribosome (>2.5 MDa) 4 . In the case of RPA (116 kDa) and many other multi-domain proteins, modularity and interdomain flexibility are the critical properties that enable characterization of dynamic architectures by NMR.To illustrate the analytical framework, results are presented first for RPA70NAB (M r 45.8 kDa), which has an asymmetric arrangement with a 70-residue N-A linker and a 10-residue A-B linker (Figure 1). The 15 N-1 H TROSY-HSQC spectrum of 15 N-enriched RPA70NAB reveals the presence of over 370 of the 400 expected signals from 422 residues (Figure 2). The signals from each of the three domains appear in positions remarkably similar to those in NMR spectra of the three isolated domains ( Figure S1). Thus, all three domains are structurally independent and resonance assignments can be transferred from the isolated domains to RPA70NAB 5 . NMR is highly sensitive to differences in the degree of inter-domain flexibility; the signals from the A and B domains are substantially weaker than the signals from the N domain, even though all three domains are approximately the same mass ( Figure 2). The differences arise from the fact that although the A and B domains are structurally independent, the short A-B tether partially restricts their motions, whereas the much longer N-A tether e...

Poly(ADP-ribose)ylation (PARylation) by PAR polymerase 1 (PARP1) and PARylation removal by poly(ADP-ribose) glycohydrolase (PARG) critically regulate DNA damage responses; yet, conflicting reports obscure PARG biology and its impact on cancer cell resistance to PARP1 inhibitors. Here, we found that PARG expression is upregulated in many cancers. We employed chemical library screening to identify and optimize methylxanthine derivatives as selective bioavailable PARG inhibitors. Multiple crystal structures reveal how substituent positions on the methylxanthine core dictate binding modes and inducible-complementarity with a PARG-specific tyrosine clasp and arginine switch, supporting inhibitor specificity and a competitive inhibition mechanism. Cell-based assays show selective PARG inhibition and PARP1 hyperPARylation. Moreover, our PARG inhibitor sensitizes cells to radiation-induced DNA damage, suppresses replication fork progression and impedes cancer cell survival. In PARP inhibitor-resistant A172 glioblastoma cells, our PARG inhibitor shows comparable killing to Nedaplatin, providing further proof-of-concept that selectively inhibiting PARG can impair cancer cell survival.

Background:The DNA repair scaffold XRCC1 binds to poly(ADP-ribose)ylated PARP1 at damaged chromatin. Results: XRCC1 preferentially binds to poly(ADP-ribose) chains longer than 7 ADP-ribose units in length. Conclusion: We identify specific determinants of XRCC1-PARP1 complex assembly, and disassembly by PARG. Significance: Our TR-FRET assay is useful for investigating turnover of posttranslational modifications and for identifying inhibitors by high-throughput screening.

Replication Protein A (RPA) is the primary eukaryotic ssDNA binding protein utilized in diverse DNA transactions in the cell. RPA is a heterotrimeric protein with seven globular domains connected by flexible linkers, which enable substantial inter-domain motion that is essential to its function. Small angle X-ray scattering (SAXS) experiments on two multi-domain constructs from the Nterminus of the large subunit (RPA70) were used to examine the structural dynamics of these domains and their response to the binding of ssDNA. The SAXS data combined with molecular dynamics simulations reveal substantial interdomain flexibility for both RPA70AB (the tandem high affinity ssDNA binding domains A and B connected by a 10-residue linker) and RPA70NAB (RPA70AB extended by a 70-residue linker to the RPA70N protein interaction domain). Binding of ssDNA to RPA70NAB reduces the interdomain flexibility between the A and B domains, but has no effect on RPA70N. These studies provide the first direct measurements of changes in orientation of these three RPA domains upon binding ssDNA. The results support a model in which RPA70N remains structurally independent of RPA70AB in the DNA bound state and therefore freely available to serve as a protein recruitment module.RPA is the primary eukaryotic ssDNA binding protein utilized for diverse DNA transactions in the replication and maintenance of the genome (reviewed by Fanning and coworkers [1]). RPA functions by binding and protecting ssDNA from degradation by endonucleases, inhibiting formation of ssDNA secondary structure, and providing a scaffold for DNA processing machinery by interacting with numerous DNA processing proteins. RPA biochemical functions and biological activities have been intensively investigated and the structures of its domains determined [2][3][4][5][6][7][8][9]. Despite this detailed information, the mechanisms for RPA function remain poorly understood, largely due to the inherent difficulties of *To whom correspondence should be addressed: Center for Structural Biology, Vanderbilt University, 465 21 st Avenue, Suite 5140, Nashville, Telephone: (615) 936-2210; Fax: (615) 936-2211; walter.chazin@vanderbilt.edu.• These authors contributed equally to this work.Experimental and theoretical scattering profiles, P(r) functions, SAXS envelopes and atomic models will be deposited in the BIOISIS database (www.bioisis.net) under accession code 61. SUPPORTING INFORMATION AVAILABLE. SEC profiles and Guinier analysis for RPA70AB, RPA70NAB and their ssDNA complexes. This material is available free of charge via the internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2011 April 6. Published in final edited form as:Biochemistry. 2010 April 6; 49(13): 2880-2889. doi:10.1021/bi9019934. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript characterizing proteins with modular organization and the fact that RPA function is integrated within complex multi-protein machinery.RPA is a mod...

SUMMARY Apoptosis-inducing factor (AIF) is critical for mitochondrial respiratory complex biogenesis and for mediating necroptotic parthanatos: these functions are seemingly regulated by enigmatic allosteric switching driven by NADH charge-transfer complex (CTC) formation. Here we define molecular pathways linking AIF’s active site to allosteric switching regions by characterizing dimer-permissive mutants using small-angle X-ray scattering (SAXS) and crystallography and by probing AIF-CTC communication networks using molecular dynamics simulations. Collective results identify two pathways propagating allostery from the CTC active site: 1) active site H454 links to S480 of AIF’s central β-strand to modulate a hydrophobic border at the dimerization interface and 2) an interaction network links AIF’s FAD cofactor, central β-strand, and Cβ-clasp whereby R529 reorientation initiates C-loop release during CTC formation. This knowledge of AIF allostery and its flavoswitch mechanism provides a foundation for biologically understanding and biomedically controlling its participation in mitochondrial homeostasis and cell death.