Bennett Van Houten - Academia.edu (original) (raw)

Papers by Bennett Van Houten

Nature Communications, Jan 6, 2015

The xeroderma pigmentosum C (XPC) complex initiates nucleotide excision repair by recognizing DNA... more The xeroderma pigmentosum C (XPC) complex initiates nucleotide excision repair by recognizing DNA lesions before recruiting downstream factors. How XPC detects structurally diverse lesions embedded within normal DNA is unknown. Here we present a crystal structure that captures the yeast XPC orthologue (Rad4) on a single register of undamaged DNA. The structure shows that a disulphide-tethered Rad4 flips out normal nucleotides and adopts a conformation similar to that seen with damaged DNA. Contrary to many DNA repair enzymes that can directly reject non-target sites as structural misfits, our results suggest that Rad4/XPC uses a kinetic gating mechanism whereby lesion selectivity arises from the kinetic competition between DNA opening and the residence time of Rad4/XPC per site. This mechanism is further supported by measurements of Rad4-induced lesion-opening times using temperature-jump perturbation spectroscopy. Kinetic gating may be a general mechanism used by site-specific DNA-binding proteins to minimize time-consuming interrogations of non-target sites.

Proceedings of the National Academy of Sciences, 2018

The version in the Kent Academic Repository may differ from the final published version. Users ar... more The version in the Kent Academic Repository may differ from the final published version. Users are advised to check http://kar.kent.ac.uk for the status of the paper. Users should always cite the published version of record.

Nature, 2016

Cell reports, Jan 25, 2014

ARTD1 (PARP1) is a key enzyme involved in DNA repair through the synthesis of poly(ADP-ribose) (P... more ARTD1 (PARP1) is a key enzyme involved in DNA repair through the synthesis of poly(ADP-ribose) (PAR) in response to strand breaks, and it plays an important role in cell death following excessive DNA damage. ARTD1-induced cell death is associated with NAD(+) depletion and ATP loss; however, the molecular mechanism of ARTD1-mediated energy collapse remains elusive. Using real-time metabolic measurements, we compared the effects of ARTD1 activation and direct NAD(+) depletion. We found that ARTD1-mediated PAR synthesis, but not direct NAD(+) depletion, resulted in a block to glycolysis and ATP loss. We then established a proteomics-based PAR interactome after DNA damage and identified hexokinase 1 (HK1) as a PAR binding protein. HK1 activity is suppressed following nuclear ARTD1 activation and binding by PAR. These findings help explain how prolonged activation of ARTD1 triggers energy collapse and cell death, revealing insight into the importance of nucleus-to-mitochondria communicat...

Nucleic Acids Research, 2013

The Saccharomyces cerevisiae Shu complex, consisting of Shu1, Shu2, Csm2 and Psy3, promotes error... more The Saccharomyces cerevisiae Shu complex, consisting of Shu1, Shu2, Csm2 and Psy3, promotes error-free homologous recombination (HR) by an unknown mechanism. Recent structural analysis of two Shu proteins, Csm2 and Psy3, has revealed that these proteins are Rad51 paralogues and mediate DNA binding of this complex. We show in vitro that the Csm2-Psy3 heterodimer preferentially binds synthetic forked DNA or 3 0-DNA overhang substrates resembling structures used during HR in vivo. We find that Csm2 interacts with Rad51 and the Rad51 paralogues, the Rad55-Rad57 heterodimer and that the Shu complex functions in the same epistasis group as Rad55-Rad57. Importantly, Csm2's interaction with Rad51 is dependent on Rad55, whereas Csm2's interaction with Rad55 occurs independently of Rad51. Consistent with the Shu complex containing Rad51 paralogues, the methyl methanesulphonate sensitivity of Csm2 is exacerbated at colder temperatures. Furthermore, Csm2 and Psy3 are needed for efficient recruitment of Rad55 to DNA repair foci after DNA damage. Finally, we observe that the Shu complex preferentially promotes Rad51-dependent homologous recombination over Rad51-independent repair. Our data suggest a model in which Csm2-Psy3 recruit the Shu complex to HR substrates, where it interacts with Rad51 through Rad55-Rad57 to stimulate Rad51 filament assembly and stability, promoting error-free repair.

Biophysical Journal, 2011

Biophysical Journal, 2013

Scientific reports, Jan 22, 2015

In this study we describe a new methodology to physically probe individual complexes formed betwe... more In this study we describe a new methodology to physically probe individual complexes formed between proteins and DNA. By combining nanoscale, high speed physical force measurement with sensitive fluorescence imaging we investigate the complex formed between the prokaryotic DNA repair protein UvrA2 and DNA. This approach uses a triangular, optically-trapped "nanoprobe" with a nanometer scale tip protruding from one vertex. By scanning this tip along a single DNA strand suspended between surface-bound micron-scale beads, quantum-dot tagged UvrA2 molecules bound to these '"DNA tightropes" can be mechanically interrogated. Encounters with UvrA2 led to deflections of the whole nanoprobe structure, which were converted to resistive force. A force histogram from all 144 detected interactions generated a bimodal distribution centered on 2.6 and 8.1 pN, possibly reflecting the asymmetry of UvrA2's binding to DNA. These observations successfully demonstrate the use...

UvrB, a central DNA damage recognition protein in bacterial nucleotide excision repair, has weak ... more UvrB, a central DNA damage recognition protein in bacterial nucleotide excision repair, has weak affinity for DNA, and its ATPase activity is activated by UvrA and damaged DNA. Regulation of DNA binding and ATP hydrolysis by UvrB is poorly understood. Using atomic force microscopy and biochemical assays, we found that truncation of domain 4 of Bacillus caldotenax UvrB (UvrB⌬4) leads to multiple changes in protein function. Protein dimerization decreases with an ϳ8-fold increase of the equilibrium dissociation constant and an increase in DNA binding. Loss of domain 4 causes the DNA binding mode of UvrB to change from dimer to monomer, and affinity increases with the apparent dissociation constants on nondamaged and damaged single-stranded DNA decreasing 22-and 14-fold, respectively. ATPase activity by UvrB⌬4 increases 14-and 9-fold with and without single-stranded DNA, respectively, and UvrB⌬4 supports UvrA-independent damage-specific incision by Cho on a bubble DNA substrate. We propose that other than its previously discovered role in regulating protein-protein interactions, domain 4 is an autoinhibitory domain regulating the DNA binding and ATPase activities of UvrB. Nucleotide excision repair (NER) 3 is a DNA repair pathway conserved from bacteria to eukaryotes. DNA damage recognition and incision during NER in prokaryotes is a complex process involving UvrA, UvrB, and UvrC (1-3). UvrA and UvrB interact in solution, forming a UvrAB complex (4). It is believed that UvrA, as a dimer within the UvrAB complex, first recognizes helical distortions induced by DNA damage. Upon binding of UvrAB (either as UvrA 2 B or UvrA 2 B 2) to the site of DNA damage (4, 5), conformational changes in the UvrAB-DNA complex lead to transfer of DNA from UvrA to UvrB and dissociation of UvrA (6-10). After dissociation of UvrA from the protein-DNA complex, a very stable UvrB-DNA preincision complex is formed. UvrC recruitment by the UvrB-DNA precincision complex to the site of DNA damage leads to dual incisions on the damaged DNA strand (11-13). UvrB plays a central role in bacterial NER. In the absence of UvrA,

Journal of Biological Chemistry, 1988

Escherichia coli ABC excinuclease initiates the removal of dodecanucleotides from damaged DNA in ... more Escherichia coli ABC excinuclease initiates the removal of dodecanucleotides from damaged DNA in an ATP-dependent reaction. Using a synthetic DNA fragment containing a psoralen adduct at a defined position we have investigated the interaction of the components of the enzyme with substrate by DNase I footprinting. We find that the UvrA subunit binds to DNA specifically in the absence of cofactors and that the binding affinity is stimulated about 4-fold by ATP and only marginally inhibited by ADP. The UvrA-DNA complexes formed in the absence of co-factors or in the presence of either ATP or ADP are remarkably similar. In contrast, adenosine 5'-0-(thiotriphosphate) increases nonspecific binding and completely abolishes the UvrA footprint. The UvrB subunit can associate with the UvrA subunit on DNA in the absence of ATP, but this ternary UvrA-UvrB-DNA complex is qualitatively different from that formed in the presence of ATP. The UvrC subunit elicits no additional change in the UvrA-UvrB footprint. Helicase I1 (UvrD protein) does not alter the UvrA-UvrB footprint but does appear to interact at the 6'-incision site of the postincision complex. DNA polymerase I fills in the excision gap in the presence or absence of helicase I1 and apparently releases the ABC excinuclease from the repaired DNA. Nearly 90% of the repair patches are 12 nucleotides long, and this length is not affected by helicase 11. We see no evidence by DNase I footprinting for the formation of a multiprotein complex encompassing the UvrA,-B,-C, and-D proteins and DNA polymerase I.

DNA Repair, 2020

UV-damaged DNA binding protein (UV-DDB) is a heterodimeric complex, composed of DDB1 and DDB2, an... more UV-damaged DNA binding protein (UV-DDB) is a heterodimeric complex, composed of DDB1 and DDB2, and is involved in global genome nucleotide excision repair. Mutations in DDB2 are associated with xeroderma pigmentosum complementation group E. UV-DDB forms a ubiquitin E3 ligase complex with cullin-4A and RBX that helps to relax chromatin around UV-induced photoproducts through the ubiquitination of histone H2A. After providing a brief historical perspective on UV-DDB, we review our current knowledge of the structure and function of this intriguing repair protein. Finally, this article discusses emerging data suggesting that UV-DDB may have other non-canonical roles in base excision repair and the etiology of cancer. 1. Introduction. Life has evolved a series of pathways to remove specific types of DNA damage. Nucleotide excision repair (NER) is dedicated to the removal of a wide variety of helix distorting lesions, including: ultraviolet (UV) photolesions, cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts (6-4PP); bulky adducts formed by chemical carcinogens, such as polycyclic aromatic hydrocarbons; and certain DNA lesions formed from chemotherapeutic agents, like cisplatin. NER consists of two sub-pathways: global genome NER (GG-NER) #

ChemBioChem, 2017

Nucleotide excision repair (NER) is a general DNA repair mechanism that is capable of removing a ... more Nucleotide excision repair (NER) is a general DNA repair mechanism that is capable of removing a wide variety of DNA lesions induced by physical or chemical insults. UvrD, a member of the helicase SF1 superfamily, plays an essential role in bacterial NER by unwinding the duplex DNA in the 3′ to 5′ direction to displace the lesion‐containing strand. In order to achieve conditional control over NER, we generated a light‐activated DNA helicase. This was achieved through a site‐specific incorporation of a genetically encoded hydroxycoumarin lysine at a crucial position in the ATP‐binding pocket of UvrD. The resulting caged enzyme was completely inactive in several functional assays. Moreover, enzymatic activity of the optically triggered UvrD was comparable to that of the wild‐type protein, thus demonstrating excellent OFF to ON switching of the helicase. The developed approach provides optical control of NER, thereby laying a foundation for the regulation of ATP‐dependent helicase func...

PLoS genetics, 2013

Stringent steric exclusion mechanisms limit the misincorporation of ribonucleotides by high-fidel... more Stringent steric exclusion mechanisms limit the misincorporation of ribonucleotides by high-fidelity DNA polymerases into genomic DNA. In contrast, low-fidelity Escherichia coli DNA polymerase V (pol V) has relatively poor sugar discrimination and frequently misincorporates ribonucleotides. Substitution of a steric gate tyrosine residue with alanine (umuC_Y11A) reduces sugar selectivity further and allows pol V to readily misincorporate ribonucleotides as easily as deoxynucleotides, whilst leaving its poor base-substitution fidelity essentially unchanged. However, the mutability of cells expressing the steric gate pol V mutant is very low due to efficient repair mechanisms that are triggered by the misincorporated rNMPs. Comparison of the mutation frequency between strains expressing wild-type and mutant pol V therefore allows us to identify pathways specifically directed at ribonucleotide excision repair (RER). We previously demonstrated that rNMPs incorporated by umuC_Y11A are eff...

DNA Repair, 2014

A powerful new approach has become much more widespread and offers insights into aspects of DNA r... more A powerful new approach has become much more widespread and offers insights into aspects of DNA repair unattainable with billions of molecules. Single molecule techniques can be used to image, manipulate or characterize the action of a single repair protein on a single strand of DNA. This allows search mechanisms to be probed, and the effects of force to be understood. These physical aspects can dominate a biochemical reaction, where at the ensemble level their nuances are obscured. In this paper we discuss some of the many technical advances that permit study at the single molecule level. We focus on DNA repair to which these techniques are actively being applied. DNA repair is also a process that encompasses so much of what single molecule studies benefit-searching for targets, complex formation, sequential biochemical reactions and substrate hand-off to name just a few. We discuss how single molecule biophysics is poised to transform our understanding of biological systems, in particular DNA repair.

Proceedings of the National Academy of Sciences, 2002