Human XPG nuclease structure, assembly, and activities with insights for neurodegeneration and cancer from pathogenic mutations.

Xeroderma pigmentosum group G (XPG) protein is both a functional partner in multiple DNA damage responses (DDR) and a pathway coordinator and structure-specific endonuclease in nucleotide excision repair (NER). Different mutations in the XPG gene ERCC5 lead to either of two distinct human diseases: Cancer-prone xeroderma pigmentosum (XP-G) or the fatal neurodevelopmental disorder Cockayne syndrome (XP-G/CS). To address the enigmatic structural mechanism for these differing disease phenotypes and for XPG's role in multiple DDRs, here we determined the crystal structure of human XPG catalytic domain (XPGcat), revealing XPG-specific features for its activities and regulation. Furthermore, XPG DNA binding elements conserved with FEN1 superfamily members enable insights on DNA interactions. Notably, all but one of the known pathogenic point mutations map to XPGcat, and both XP-G and XP-G/CS mutations destabilize XPG and reduce its cellular protein levels. Mapping the distinct mutation classes provides structure-based predictions for disease phenotypes: Residues mutated in XP-G are positioned to reduce local stability and NER activity, whereas residues mutated in XP-G/CS have implied long-range structural defects that would likely disrupt stability of the whole protein, and thus interfere with its functional interactions. Combined data from crystallography, biochemistry, small angle X-ray scattering, and electron microscopy unveil an XPG homodimer that binds, unstacks, and sculpts duplex DNA at internal unpaired regions (bubbles) into strongly bent structures, and suggest how XPG complexes may bind both NER bubble junctions and replication forks. Collective results support XPG scaffolding and DNA sculpting functions in multiple DDR processes to maintain genome stability.

Thirdhand smoke exposure causes replication stress and impaired transcription in human lung cells.

Thirdhand cigarette smoke (THS) is a newly described toxin that lingers in the indoor environment long after cigarettes have been extinguished. Emerging results from both cellular and animal model studies suggest that THS is a potential human health hazard. DNA damage derived from THS exposure could have genotoxic consequences that would lead to the development of diseases. However, THS exposure-induced interference with fundamental DNA transactions such as replication and transcription, and the role of DNA repair in ameliorating such effects, remain unexplored. Here, we found that THS exposure increased the percentage of cells in S-phase, suggesting impaired S-phase progression. Key DNA damage response proteins including RPA, ATR, ATM, CHK1, and BRCA1 were activated in lung cells exposed to THS, consistent with replication stress. In addition, THS exposure caused increased 53BP1 foci, indicating DNA double-strand break induction. Consistent with these results, we observed increased micronuclei formation, a marker of genomic instability, in THS-exposed cells. Exposure to THS also caused a significant increase in phosphorylated RNA Polymerase II engaged in transcription elongation, suggesting an increase in transcription-blocking lesions. In agreement with this conclusion, ongoing RNA synthesis was very significantly reduced by THS exposure. Loss of nucleotide excision repair exacerbated the reduction in RNA synthesis, suggesting that bulky DNA adducts formed by THS are blocks to transcription. The adverse impact on both replication and transcription supports genotoxic stress as a result of THS exposure, with important implications for both cancer and other diseases.

Overexpression of the base excision repair NTHL1 glycosylase causes genomic instability and early cellular hallmarks of cancer.

Base excision repair (BER), which is initiated by DNA N-glycosylase proteins, is the frontline for repairing potentially mutagenic DNA base damage. The NTHL1 glycosylase, which excises DNA base damage caused by reactive oxygen species, is thought to be a tumor suppressor. However, in addition to NTHL1 loss-of-function mutations, our analysis of cancer genomic datasets reveals that NTHL1 frequently undergoes amplification or upregulation in some cancers. Whether NTHL1 overexpression could contribute to cancer phenotypes has not yet been explored. To address the functional consequences of NTHL1 overexpression, we employed transient overexpression. Both NTHL1 and a catalytically-dead NTHL1 (CATmut) induce DNA damage and genomic instability in non-transformed human bronchial epithelial cells (HBEC) when overexpressed. Strikingly, overexpression of either NTHL1 or CATmut causes replication stress signaling and a decrease in homologous recombination (HR). HBEC cells that overexpress NTHL1 or CATmut acquire the ability to grow in soft agar and exhibit loss of contact inhibition, suggesting that a mechanism independent of NTHL1 catalytic activity contributes to acquisition of cancer-related cellular phenotypes. We provide evidence that NTHL1 interacts with the multifunctional DNA repair protein XPG suggesting that interference with HR is a possible mechanism that contributes to acquisition of early cellular hallmarks of cancer.

Non-catalytic Roles for XPG with BRCA1 and BRCA2 in Homologous Recombination and Genome Stability.

XPG is a structure-specific endonuclease required for nucleotide excision repair, and incision-defective XPG mutations cause the skin cancer-prone syndrome xeroderma pigmentosum. Truncating mutations instead cause the neurodevelopmental progeroid disorder Cockayne syndrome, but little is known about how XPG loss results in this devastating disease. We identify XPG as a partner of BRCA1 and BRCA2 in maintaining genomic stability through homologous recombination (HRR). XPG depletion causes DNA double-strand breaks, chromosomal abnormalities, cell-cycle delays, defective HRR, inability to overcome replication fork stalling, and replication stress. XPG directly interacts with BRCA2, RAD51, and PALB2, and XPG depletion reduces their chromatin binding and subsequent RAD51 foci formation. Upstream in HRR, XPG interacts directly with BRCA1. Its depletion causes BRCA1 hyper-phosphorylation and persistent chromatin binding. These unexpected findings establish XPG as an HRR protein with important roles in genome stability and suggest how XPG defects produce severe clinical consequences including cancer and accelerated aging.

The DNA repair endonuclease XPG interacts directly and functionally with the WRN helicase defective in Werner syndrome.

XPG is a structure-specific endonuclease required for nucleotide excision repair (NER). XPG incision defects result in the cancer-prone syndrome xeroderma pigmentosum, whereas truncating mutations of XPG cause the severe postnatal progeroid developmental disorder Cockayne syndrome. We show that XPG interacts directly with WRN protein, which is defective in the premature aging disorder Werner syndrome, and that the two proteins undergo similar subnuclear redistribution in S phase and colocalize in nuclear foci. The co-localization was observed in mid- to late S phase, when WRN moves from nucleoli to nuclear foci that have been shown to contain both protein markers of stalled replication forks and telomeric proteins. We mapped the interaction between XPG and WRN to the C-terminal domains of each, and show that interaction with the C-terminal domain of XPG strongly stimulates WRN helicase activity. WRN also possesses a competing DNA single-strand annealing activity that, combined with unwinding, has been shown to coordinate regression of model replication forks to form Holliday junction/chicken foot intermediate structures. We tested whether XPG stimulated WRN annealing activity, and found that XPG itself has intrinsic strand annealing activity that requires the unstructured R- and C-terminal domains but not the conserved catalytic core or endonuclease activity. Annealing by XPG is cooperative, rather than additive, with WRN annealing. Taken together, our results suggest a novel function for XPG in S phase that is, at least in part, performed coordinately with WRN, and which may contribute to the severity of the phenotypes that occur upon loss of XPG.

Pre-steady-state binding of damaged DNA by XPC-hHR23B reveals a kinetic mechanism for damage discrimination.

The XPC-hHR23B complex (XPC-hHR23B) is a heterodimeric protein required for the initial step of DNA damage recognition in the global nucleotide excision repair (NER) pathway. A strong preference of XPC-hHR23B for UV- and cisplatin-damaged DNA has previously been demonstrated using equilibrium binding assays. To better understand the molecular mechanism of damage recognition by XPC-hHR23B, we carried out the pre-steady-state kinetic analysis of the XPC-hHR23B-DNA interactions using a stopped-flow fluorescence assay. XPC-hHR23B displays a faster k(on) for cisplatin- and UV-damaged duplex DNA than for undamaged DNA, with additional, minor effects on the k(off) rates. XPC-hHR23B has a high affinity for undamaged single-stranded DNA compared to duplex DNA, which can be largely attributed to a high rate of association. However, cisplatin damage on single-stranded DNA reduced the overall level of binding by a factor of 7, with nearly equal contributions from changes to the k(on) and k(off) rates. Together, these results support a model for initial damage recognition by XPC-hHR23B that is dependent on structural changes in the DNA, and not adduct chemistry.

Cisplatin sensitizes cancer cells to ionizing radiation via inhibition of nonhomologous end joining.

The combination of cisplatin and ionizing radiation (IR) treatment represents a common modality for treating a variety of cancers. These two agents provide considerable synergy during treatment, although the mechanism of this synergy remains largely undefined. We have investigated the mechanism of cisplatin sensitization to IR using a combination of in vitro and in vivo experiments. A clear synergistic interaction between cisplatin and IR is observed in cells proficient in nonhomologous end joining (NHEJ) catalyzed repair of DNA double-strand breaks (DSB). In contrast, no interaction between cisplatin and IR is observed in NHEJ-deficient cells. Reconstituted in vitro NHEJ assays revealed that a site-specific cisplatin-DNA lesion near the terminus results in complete abrogation of NHEJ catalyzed repair of the DSB. These data show that the cisplatin-IR synergistic interaction requires the DNA-dependent protein kinase-dependent NHEJ pathway for joining of DNA DSBs, and the presence of a cisplatin lesion on the DNA blocks this pathway. In the absence of a functional NHEJ pathway, although the cells are hypersensitive to IR, there is no synergistic interaction with cisplatin.

The herpes simplex virus type 1 DNA polymerase processivity factor, UL42, does not alter the catalytic activity of the UL9 origin-binding protein but facilitates its loading onto DNA.

The herpes simplex virus type 1 UL42 DNA polymerase processivity factor interacts physically with UL9 and enhances its ability to unwind short, partially duplex DNA. In this report, ATP hydrolysis during translocation of UL9 on single-stranded (ss) or partially duplex DNA was examined in the presence and absence of UL42 to determine the effect of UL42 on the catalytic function of UL9. Our studies reveal that a homodimer of UL9 is sufficient for DNA translocation coupled to ATP hydrolysis, and the steady-state ATPase catalytic rate was greater on partially duplex DNA than on ss DNA in the presence or absence of UL42. Although UL42 protein increased the steady-state rate for ATP hydrolysis by UL9 during translocation on either partially duplex or ss DNA, UL42 had no significant effect on the intrinsic ATPase activity of UL9. UL42 also had no effect on the catalytic rate of ATP hydrolysis when UL9 was not limiting but enhanced the steady-state ATPase rate at only subsaturating UL9 concentrations. At subsaturating UL9 to DNA ratios, stoichiometric concentrations of UL42 were shown to increase the amount of UL9 bound to ss DNA at equilibrium. These data support a model whereby UL42 increases the ability of UL9 to load onto DNA, thus increasing its ability to assemble into a functional complex capable of unwinding duplex DNA.

Functional interaction between the herpes simplex virus type 1 polymerase processivity factor and origin-binding proteins: enhancement of UL9 helicase activity.

The origin (ori)-binding protein of herpes simplex virus type 1 (HSV-1), encoded by the UL9 open reading frame, has been shown to physically interact with a number of cellular and viral proteins, including three HSV-1 proteins (ICP8, UL42, and UL8) essential for ori-dependent DNA replication. In this report, it is demonstrated for the first time that the DNA polymerase processivity factor, UL42 protein, provides accessory function to the UL9 protein by enhancing the 3'-to-5' helicase activity of UL9 on partially duplex nonspecific DNA substrates. UL42 fails to enhance the unwinding activity of a noncognate helicase, suggesting that enhancement of unwinding requires the physical interaction between UL42 and UL9. UL42 increases the steady-state rate for unwinding a 23/38-mer by UL9, but only at limiting UL9 concentrations, consistent with a role in increasing the affinity of UL9 for DNA. Optimum enhancement of unwinding was observed at UL42/UL9 molecular ratios of 4:1, although enhancement was reduced when high UL42/DNA ratios were present. Under the assay conditions employed, UL42 did not alter the rate constant for dissociation of UL9 from the DNA substrate. UL42 also did not significantly reduce the lag period which was observed following the addition of UL9 to DNA, regardless of whether UL42 was added to DNA prior to or at the same time as UL9. Moreover, addition of UL42 to ongoing unwinding reactions increased the steady-state rate for unwinding, but only after a 10- to 15-min lag period. Thus, the increased affinity of UL9 for DNA most likely is the result of an increase in the rate constant for binding of UL9 to DNA, and it explains why helicase enhancement is observed only at subsaturating concentrations of UL9 with respect to DNA. In contrast, ICP8 enhances unwinding at both saturating and subsaturating UL9 concentrations and reduces or eliminates the lag period. The different means by which ICP8 and UL42 enhance the ability of UL9 to unwind DNA suggest that these two members of the presumed functional replisome may act synergistically on UL9 to effect initiation of HSV-1 DNA replication in vivo.

3' to 5' exonuclease activity of herpes simplex virus type 1 DNA polymerase modulates its strand displacement activity.

Using a minicircle DNA primer-template, the wild-type catalytic subunit of herpes simplex virus type 1 (HSV-1) DNA polymerase (pol) was shown to lack significant strand displacement activity with or without its processivity factor, UL42. However, an exonuclease-deficient (exo(-)) pol (D368A) was capable of slow strand displacement. Although UL42 increased the rate (2/s) and processivity of strand displacement by exo(-) pol, the rate was slower than that for gap-filling synthesis. High inherent excision rates on matched primer-templates and rapid idling-turnover (successive rounds of excision and polymerization) of exo-proficient polymerases correlated with poor strand displacement activity. The results suggest that the exo activity of HSV-1 pol modulates its ability to engage in strand displacement, a function that may be important to the viability and genome stability of the virus.