Shurong Huang

The premature ageing syndrome protein, WRN, is a 3'-->5' exonuclease.

Werner syndrome (WS) is a human autosomal recessive disorder that causes the premature appearance of a partial array of disorders characteristic of old age1,2. These disorders include atherosclerosis, cancer, type 2 diabetes, osteoporosis, cataracts, wrinkled skin and grey hair, among other ailments. Cells cultured from WS subjects have a shortened replicative life span3,4 and elevated rates of chromosome translocations, large deletions and homologous recombination5,6. The gene defective in WS, WRN, encodes a large RecQ-like DNA helicase7 of 1432 aa. Defects in another human RecQ-like helicase, BLM, result in Bloom’s syndrome8 (BS), a genetic disorder that is quite different from WS. BS is manifested by short stature, neoplasia, immunodeficiency and high risk of cancer. Cells from BS subjects show an increase in sister chromatid exchanges. DNA helicases can function in replication, repair, recombination, transcription or RNA processing. As WRN and BLM share no obvious homology outside the helicase domain, the non-helicase domains probably determine in which process each RecQ-like helicase participates, which provides the basis for the disparate cellular and organismal phenotypes that result from defects in these proteins. Statistical sequence analyses showed subtle but significant similarities between WRN and several 3′→5′ exonucleases9,10. To test the prediction that WRN is an exonuclease, we produced tagged recombinant wild-type and mutant WRN proteins. Two mutants had amino-acid substitutions at either position 82 (D82A) or 84 (E84A), two of the five residues predicted to be critical for exonuclease activity9,10. A third mutant had a substitution at position 577 (K577M), which abolished WRN helicase activity11. The fourth mutant was an N-terminal fragment (aa 1–333; N333) containing the putative exonuclease domain, but lacking the helicase domain. A tagged 36-aa vector-derived peptide served as a negative control (mock). Purified WRN and mock proteins were incubated with doubled-stranded DNA substrates. Wild-type WRN degraded a 5′ labelled substrate to a series of smaller, labelled products (Fig. 1a), and a 3′ labelled substrate to a single labelled product that migrated as a mononucleotide (Fig. 1b). Thus, WRN degraded DNA with 3′→5′ directionality. Although mock and full-length WRN preparations contained low levels of a contaminating 5′→3′ exonuclease, as shown by release of the 5′ label as a mononucleotide (Figs 1a,​,2b),2b), 3′→5′ degradation was entirely dependent on WRN. Fig. 1 Exonuclease activity of wild-type WRN and the N333 fragment. 6×his-tagged proteins were purified from baculovirus-infected insect cells using either nuclear (WRN, D82A, E84A, K577M, mock control) or cytosolic (N333, mock control) extracts. WRN, ... Fig. 2 Helicase and exonuclease activities of wild-type and mutant WRN proteins. a, WRN, K577M, E84A, D82A or mock proteins (10 ng) were assayed for helicase activity by incubating 5′ 32P-labelled DNA substrate (0.4 pmol; Fig. 1a) in helicase assay buffer ... WRN exonuclease activity resided in the N terminus. N333, which was essentially free of contaminating 5′→3′ exonuclease, degraded 5′ and 3′ labelled substrates similarly to full-length WRN (Fig. 1c,d). When incubated with a 374-bp DNA fragment labelled at the 3′ end with 32P, and internally with 3H, N333 released most of both labels (Fig. 1e). Thus, the WRN exonuclease is capable of substantial DNA degradation. Consistent with 3′→5′ directionality, N333 released 32P from 3′ ends more rapidly than 3H from internal residues. In addition, gel-purified N333, which lacked contaminating nuclease activities, efficiently removed the 3′, but not the 5′, label when incubated with DNA substrates labelled at either the 3′ or the 5′ end (Fig. 1f). Genetic evidence for WRN exonuclease activity was obtained by introducing point mutations at critical amino acids in the exonuclease domain (D82A and E84A). These mutants retained the wild-type level of helicase activity (Fig. 2a), but had little or no 3′→5′ exonuclease activity, using either a 5′ (Fig. 2b) or 3′ (Fig. 2c) 32P-labelled substrate, and were indistinguishable from mock protein in this regard (Fig. 2d). The K577M mutant, in contrast, was devoid of helicase activity (Fig. 2a), as expected, but had 3′→5′ exonuclease activity comparable to that of wild-type WRN (Fig. 2b–d). Our data indicate that WRN is indeed a 3′→5′ exonuclease. This activity resides in the N terminus, and is physically and functionally separable from the helicase activity. The identification of an exonuclease activity in WRN clearly distinguishes it from other human RecQ-like helicases, and may help explain the differences between WS and BS. What are the functions of the WRN exonuclease in vivo? It may participate in recombination and DNA repair. Exonucleases are integral components of some recombination pathways12, and WRN appears to have a role in recombination5,6,13. The finding that WS cells are hypersensitive to the DNA damaging agent 4-nitroquinoline-1-oxide14 suggests a role for WRN in DNA repair. Finally, WRN is homologous to FFA-1 (replication focus-forming activity 1) in Xenopus laevis15, raising the possibility that WRN may also be involved in DNA replication. In this context, the WRN exonuclease may provide 3′→5′ proofreading function to DNA polymerases that lack such activity. Whatever the case, an understanding of the functions of WRN exonuclease and their relationships to the other functions of WRN will lead to new insights into the molecular and cellular basis for WS and a subset of age-associated pathologies.

WRN, the protein deficient in Werner syndrome, plays a critical structural role in optimizing DNA repair.

Werner syndrome (WS) predisposes patients to cancer and premature aging, owing to mutations in WRN. The WRN protein is a RECQ‐like helicase and is thought to participate in DNA double‐strand break (DSB) repair by non‐homologous end joining (NHEJ) or homologous recombination (HR). It has been previously shown that non‐homologous DNA ends develop extensive deletions during repair in WS cells, and that this WS phenotype was complemented by wild‐type (wt) WRN. WRN possesses both 3′ → 5′ exonuclease and 3′ → 5′ helicase activities. To determine the relative contributions of each of these distinct enzymatic activities to DSB repair, we examined NHEJ and HR in WS cells (WRN–/–) complemented with either wtWRN, exonuclease‐defective WRN (E–), helicase‐defective WRN (H–) or exonuclease/helicase‐defective WRN (E–H–). The single E– and H– mutants each partially complemented the NHEJ abnormality of WRN–/– cells. Strikingly, the E–H– double mutant complemented the WS deficiency nearly as efficiently as did wtWRN. Similarly, the double mutant complemented the moderate HR deficiency of WS cells nearly as well as did wtWRN, whereas the E– and H– single mutants increased HR to levels higher than those restored by either E–H– or wtWRN. These results suggest that balanced exonuclease and helicase activities of WRN are required for optimal HR. Moreover, WRN appears to play a structural role, independent of its enzymatic activities, in optimizing HR and efficient NHEJ repair. Another human RECQ helicase, BLM, suppressed HR but had little or no effect on NHEJ, suggesting that mammalian RECQ helicases have distinct functions that can finely regulate recombination events.

Collaboration of Werner syndrome protein and BRCA1 in cellular responses to DNA interstrand cross-links.

Cells deficient in the Werner syndrome protein (WRN) or BRCA1 are hypersensitive to DNA interstrand cross-links (ICLs), whose repair requires nucleotide excision repair (NER) and homologous recombination (HR). However, the roles of WRN and BRCA1 in the repair of DNA ICLs are not understood and the molecular mechanisms of ICL repair at the processing stage have not yet been established. This study demonstrates that WRN helicase activity, but not exonuclease activity, is required to process DNA ICLs in cells and that WRN cooperates with BRCA1 in the cellular response to DNA ICLs. BRCA1 interacts directly with WRN and stimulates WRN helicase and exonuclease activities in vitro. The interaction between WRN and BRCA1 increases in cells treated with DNA cross-linking agents. WRN binding to BRCA1 was mapped to BRCA1 452–1079 amino acids. The BRCA1/BARD1 complex also associates with WRN in vivo and stimulates WRN helicase activity on forked and Holliday junction substrates. These findings suggest that WRN and BRCA1 act in a coordinated manner to facilitate repair of DNA ICLs.