Werner syndrome (WS) is an adult onset segmental progeroid syndrome caused by mutations in the WRN gene. The WRN gene encodes a 180 kDa nuclear protein that possesses helicase and exonuclease activities. The absence of WRN protein leads to abnormalities in various DNA metabolic pathways such as DNA repair, replication and telomere maintenance. Individuals with WS generally develop normally until the third decade of life, when premature aging phenotypes and a series of age-related disorders begin to manifest. In Japan, where a founder effect has been described, the frequency of Werner heterozygotes appears to be as high as 1/180 in the general population. Due to the relatively non-specific nature of the symptoms and the lack of awareness of the condition, this disease may be under-diagnosed in other parts of the world. Genetic counseling of WS patients follows the path of other autosomal recessive disorders, with special attention needed for cancer surveillance in relatives. Molecular diagnosis of WS is made by nucleotide sequencing and, in some cases, protein analysis. It is also of potential interest to measure WRN activities in WS patients. More than 50 different disease-causing mutations in the WRN gene have been identified in WS patients from all over the world. All but one of these cases has mutations that result in the premature termination of the protein. Here we describe the clinical, molecular and biochemical characteristics of WS for use by medical professionals in a health care setting. Additional information is available through the International Registry of WS (http://www.wernersyndrome.org).

Cockayne Syndrome (CS) is a human genetic disorder with two complementation groups, CS-A and CS-B. The CSB gene product is involved in transcription-coupled repair of DNA damage but may participate in other pathways of DNA metabolism. The present study investigated the role of different conserved helicase motifs of CSB in base excision repair. Stably transformed human cell lines with site-directed CSB mutations in different motifs within its putative helicase domain were established. We find that CSB null and helicase motif V and VI mutants had greater sensitivity than wild type cells to ␥-radiation. Whole cell extracts from CSB null and motif V/VI mutants had lower activity of 8-hydroxyguanine incision in DNA than wild type cells. Also, 8-hydroxyguanine accumulated more in CSB null and motif VI mutant cells than in wild type cells after exposure to ␥-radiation. We conclude that a deficiency in general genome base excision repair of selective modified DNA base(s) might contribute to CS pathogenesis. Furthermore, whereas the disruption of helicase motifs V or VI results in a CSB phenotype, mutations in other helicase motifs do not cause this effect. The biological functions of CSB in different DNA repair pathways may be mediated by distinct functional motifs of the protein.

Cockayne syndrome (CS) is a human premature aging disorder associated with severe developmental deficiencies and neurodegeneration, and phenotypically it resembles some mitochondrial DNA (mtDNA) diseases. Most patients belong to complementation group B, and the CS group B (CSB) protein plays a role in genomic maintenance and transcriptome regulation. By immunocytochemistry, mitochondrial fractionation, and Western blotting, we demonstrate that CSB localizes to mitochondria in different types of cells, with increased mitochondrial distribution following menadione-induced oxidative stress. Moreover, our results suggest that CSB plays a significant role in mitochondrial base excision repair (BER) regulation. In particular, we find reduced 8-oxo-guanine, uracil, and 5-hydroxy-uracil BER incision activities in CSB-deficient cells compared to wild-type cells. This deficiency correlates with deficient association of the BER activities with the mitochondrial inner membrane, suggesting that CSB may participate in the anchoring of the DNA repair complex. Increased mutation frequency in mtDNA of CSB-deficient cells demonstrates functional significance of the presence of CSB in the mitochondria. The results in total suggest that CSB plays a direct role in mitochondrial BER by helping recruit, stabilize, and/or retain BER proteins in repair complexes associated with the inner mitochondrial membrane, perhaps providing a novel basis for understanding the complex phenotype of this debilitating disorder.

The parkin protein functions as a RING-type ubiquitin protein ligase. Considering the possibility that impaired ubiquitin-proteosomal system activity may impair antioxidant defenses and enhance oxidative stress, we have investigated the activity of mitochondrial respiratory enzymes in patients with parkin gene mutations. A significant decrease in the leukocyte complex I activity was found both in patients with parkin mutations (62.5%) and idiopathic PD (64.5%) compared with age-matched controls (P < 0.001). Complex IV activity was also decreased significantly in idiopathic PD patients (60%), but no difference was detected between controls and patients with parkin mutations.

The Cockayne syndrome B (CSB) protein—defective in a majority of patients suffering from the rare autosomal disorder CS—is a member of the SWI2/SNF2 family with roles in DNA repair and transcription. We demonstrate herein that purified recombinant CSB and the major human apurinic/apyrimidinic (AP) endonuclease, APE1, physically and functionally interact. CSB stimulates the AP site incision activity of APE1 on normal (i.e. fully paired) and bubble AP–DNA substrates, with the latter being more pronounced (up to 6-fold). This activation is ATP-independent, and specific for the human CSB and full-length APE1 protein, as no CSB-dependent stimulation was observed with Escherichia coli endonuclease IV or an N-terminal truncated APE1 fragment. CSB and APE1 were also found in a common protein complex in human cell extracts, and recombinant CSB, when added back to CSB-deficient whole cell extracts, resulted in increased total AP site incision capacity. Moreover, human fibroblasts defective in CSB were found to be hypersensitive to both methyl methanesulfonate (MMS) and 5-hydroxymethyl-2′-deoxyuridine, agents that introduce base excision repair (BER) DNA substrates/intermediates.

Cockayne syndrome (CS) 6 is a segmental premature aging syndrome with progressive neurological degeneration (1). CS is caused by mutations in CS complementation groups A (CSA) or B (CSB) genes (2, 3). Approximately 80% of CS patients have mutations in the CSB gene, which encodes a 168-kDa protein belonging to the SWI/SNF2 family of chromatin remodeling proteins (4). Cells from CS patients are hypersensitive to UV radiation-induced DNA damage, and the CSB protein is required for the transcription-coupled nucleotide excision repair of UV radiation-induced DNA lesions (cyclobutane pyrimidine dimers and 6-pyrimidine-4-pyrimidone products) (5). CSB is also believed to play a role in transcription elongation and interacts with the RNA polymerase II elongation complex (6). The molecular basis of the progressive neurological defects in CS patients, however, remains unknown; it has been proposed that neurological symptoms in CS may be due to defective repair and/or processing of oxidative DNA damage in CSB-deficient cells (7).Oxidative DNA damage can be caused by endogenous and exogenous agents. Reactive oxygen species, including highly reactive hydroxyl radicals, are formed as byproducts of normal metabolism, mostly during the process of mitochondrial respiration. It has been estimated that up to 2% of all the O 2 consumed by respiration may be released as reactive oxygen species (8, 9). The central nervous system relies exclusively on mitochondria to generate ATP through oxidative metabolism. As a result, neurons are susceptible to increased levels of oxidative stress, and elevated levels of reactive oxygen species have been implicated in the etiology of neurodegenerative diseases including Alzheimer, Parkinson, and Huntington diseases and amyotrophic lateral sclerosis (for a review, see Ref. 10).Hydroxyl radicals attack DNA bases and the sugar-phosphate DNA backbone, generating modified bases and singlestranded DNA (ssDNA) breaks, respectively (11). Many oxida-

Cockayne Syndrome (CS) is a rare human genetic disorder characterized by progressive multisystem degeneration and segmental premature aging. The CS complementation group B (CSB) protein is engaged in transcription coupled and global nucleotide excision repair, base excision repair and general transcription. However, the precise molecular function of the CSB protein is still unclear. In the current review we discuss the involvement of CSB in some of these processes, with focus on the role of CSB in repair of oxidative damage, as deficiencies in the repair of these lesions may be an important aspect of the premature aging phenotype of CS.

Werner syndrome (WS) is a human genetic disorder characterized by extensive clinical features of premature aging. Ataxia-telengiectasia (A-T) is a multisystem human genomic instability syndrome that includes premature aging in some of the patients. WRN and ATM, the proteins defective in WS and A-T, respectively, play significant roles in the maintenance of genomic stability and are involved in several DNA metabolic pathways. A role for WRN in DNA repair has been proposed; however, this study provides evidence that WRN is also involved in ATM pathway activation and in a S-phase checkpoint in cells exposed to DNA interstrand cross-link-induced double-strand breaks. Depletion of WRN in such cells by RNA interference results in an intra-S checkpoint defect, and interferes with activation of ATM as well as downstream phosphorylation of ATM target proteins. Treatment of cells under replication stress with the ATM kinase inhibitor KU 55933 results in a S-phase checkpoint defect similar to that observed in WRN shRNA cells. Moreover, ␥H2AX levels are higher in WRN shRNA cells than in control cells 6 and 16 h after exposure to psoralen DNA cross-links. These results suggest that WRN and ATM participate in a replication checkpoint response, in which WRN facilitates ATM activation in cells with psoralen DNA cross-link-induced collapsed replication forks. INTRODUCTIONProliferating cells are continuously exposed to agents that cause DNA damage and/or interfere with the progression of DNA replication (i.e., induce DNA replication fork arrest). Because such events can adversely affect genomic stability, cells have evolved S-phase signaling cascades, including DNA damage checkpoint responses and DNA repair mechanisms, to remove or repair DNA damage. In cells with DNA damage, the S-phase checkpoint inhibits the firing of late origins of replication (intra-S checkpoint) and prevents cells from entering mitosis with persistent DNA lesions (S-M checkpoint; Bartek et al., 2004). Together, these mechanisms promote accurate completion of DNA replication before cell division. Cells with defective S-phase cell cycle checkpoints are characterized by high levels of genomic instability.Many commonly used cancer therapeutic agents sensitize cancer cells to replication-blocking DNA damage Farmer et al., 2005). For example, psoralen plus UVA (PUVA) induces interstrand cross-links (ICLs), which covalently bind the complementary strands of the DNA double helix, causing DNA replication fork arrest, and inducing cytotoxic DSBs during S-phase (Rothfuss and Grompe, 2004). Similarly, camptothecin (CPT) induces and traps topoisomerase I-DNA covalent complexes, stabilizing otherwise transient DNA single-strand breaks and ultimately producing cytotoxic DNA double-strand breaks (DSBs) during S-phase (Pommier et al., 2003). Exposure to hydroxyurea (HU), which depletes dNTP pools, has similar effects on cycling cells (Lundin et al., 2002). Although the mechanisms by which these clastogens act are distinct, they all indirectly induce DSBs in the vicinity of...