During replication, transcription, or recombination, DNA strands must be separated in order for proteins to access genetic information (1). This reaction is catalyzed by DNA helicases, which include the extensively studied and highly conserved RecQ family (6). Due to their role in maintaining genome stability, these genes are considered caretakers (1).
Structurally, RecQ family proteins all possess helicase and RecQ Carboxyl-terminal (RQC) domains, while displaying significant variation in their N-terminal sequences (1). The helicase domain is involved in strand separation of double-stranded DNA, while the RQC is central for energy generation through ATP hydrolysis, DNA binding, as well as the helicase function (1). In humans, five RecQ proteins have been identified: BLM, WRN, RECQ1, RECQ4 and RECQ5 (1). Mutations in three of the proteins, BLM, WRN, and RECQ4, contribute to three genetically-based syndromes: Bloom syndrome, Werner syndrome, and Rothmund-Thomson syndrome respectively.
A variety of mutations in WRN cause Werner Syndrome (WS), an autosomal recessive disease characterized by premature aging, changes in the skin, baldness, and death at the average age of 50 due to cancer or vascular diseases (7, 11). Cancers of mesenchymal origin, such as soft tissue and bone sarcomas, are often observed (1). Other signature phenotypes of the disease include type II diabetes, atherosclerosis, osteoporosis and cataracts (1). The WRN gene, located on the short arm of chromosome q8, codes for a 1432 amino acid protein with a HRDC (Helicase and RNase D C-terminal) and a N-terminal exonuclease domain in addition to the traditional helicase and RQC domains (2). Thus, WRN is mostly involved in exonuclease and helicase activities.
Nonsense, splicing, or frameshift mutations in WRN are commonly found in the genome of WS patients, and the disease itself is only observed when WRN's exonuclease and helicase functions are simultaneously lost (1). Furthermore, fibroblasts from patients show reduced proliferative lifespan, excessive chromosomal translocations, and increased genomic deletions (1). Hypersensitivity of cells in WS patients to DNA damaging agents indicates a defect in the DNA repair mechanism (3). It is hypothesized that mutations in WS result in aberrant WRN proteins, which cannot localize to the nucleus and are instead rapidly degraded (3).
Bloom’s Syndrome (BS) is an autosomal recessive disorder caused by mutations in BLM. Typically, patients display proportional dwarfism, hypersensitivity to light, immunodeficiency, and a predisposition to cancer at the average age of 24 (1, 4). In terms of the molecular profile, BS cells have high frequencies of chromosomal aberrations, hypersensitivity to DNA-damaging agents (e.g., hydroxyurea), increased number of micronuclei, and high frequency (e.g., tenfold) of sister chromatid exchanges (SCEs), which occur during homologous recombination in response to DNA damage in the S or G2 phases (1, 4).
The BLM gene codes for a 1417 amino acid helicase, which shares three domains with WRN: helicase, RQC, and HRDC(1). The RQC domain has a Zn2+-binding region with four conserved cysteine residues. Mutation in any two of these residues results in BS. The HRDC domain allows specific binding to DNA in order to unwind it (4). The N-terminal of BLM contains a repeat sequence thought to be important for interactions between BLM and other proteins (4). Post-translational modifications have been shown to impact localization and function of the BLM protein (4). Mutations in BLM result in low levels of BLM mRNA and termination of protein translation due to nonsense-mediated mRNA decay (1).
RECQ4 is also involved in DNA replication. Much less is known about how this protein works at the cellular level. Some research suggests that RECQ4 plays a role in double-strand break repair, where it may help govern which repair pathway is chosen (11). However, nonsense and frameshift mutations in the RECQ4 gene are known to contribute to the autosomal recessive Rothmund-Thomson syndrome, which is characterized by premature aging and predisposition to cancer, particularly in the bone (11). Only 60% of patients with Rothmond-Thomson syndrome harbour a mutation in the RECQ4 gene, suggesting that multiple genetic factors contribute to the disease (11).
WRN and BLM Functions
WRN and BLM are involved in several aspects of DNA metabolism including replication, repair, and telomere preservation (5). Replication errors resulting in formation of double-stranded breaks (DSBs) or broken replication forks are commonly repaired through homologous recombination (HR). As the initiating step in HR, the DSB ends must be resected or digested in a 5’ to 3’ manner, and this is promoted by BLM through stimulation of exonuclease I (4). The formation of Holliday junctions and their subsequent removal into non-crossover products is also mediated by BLM (4). An animation of double-strand break repair can be found here.
Given this finding and the fact that BS cells demonstrate high frequencies of conventional Holliday junctions, which inevitably result in 50-50 crossover rates and high frequency of SCEs, it appears that a predominant role of BLM is to minimize the frequency of crossovers (4). In rare cases, crossovers may result in altered chromosomal products with carcinogenic potential, so the presence of inhibitory processes is essential for the maintenance of chromosomal integrity in long-living diploid organisms. WRN’s role in HR involves formation of a multi-protein complex with ATR, RAD51, RAD52, RAD54 and RAD54B, although the function of WRN in this capacity is still unclear (6).
BLM’s role in DNA replication involves the removal of DNA tertiary structures, including hairpins and G-quadruplexes, that form after strand separation. Left alone, these structures would complicate and arrest the replication process (1). In cases where replication fork stalling occurs, several outcomes are possible. Generally, BLM suppresses the activity of multiple dormant replication origins that will otherwise open to compensate for a slower replication rate (4). Under certain circumstances, BLM and WRN promote the regression of the stalled fork, where nascent strands remove the template strands and anneal to one another (4). In DNA replication, WRN assists in synthesis of the telomere lagging strand. During the synthesis of the telomere lagging strand, WRN associates with the TTAGGG telomeric region, unwinding G-quadruplexes and allowing replication forks to continue to the chromosomal ends (4). Co-localization of WRN with ATR and S-phase checkpoint proteins in response to replication stress also suggests a role for this protein in replication checkpoint regulation (5).
1. Chu, W. K., & Hickson, I. D. (2009). RecQ helicases: multifunctional genome caretakers. Nature Reviews Cancer, 9(9), 644-654.
2. Nakayama, H. (2002). RecQ family helicases: roles as tumor suppressor proteins. Oncogene, 21(58).
3. Li, B., Navarro, S., Kasahara, N., & Comai, L. (2004). Identification and biochemical characterization of a Werner's syndrome protein complex with Ku70/80 and poly (ADP-ribose) polymerase-1. Journal of Biological Chemistry,279(14), 13659-13667.
4. Payne, M., & Hickson, I. D. (2009). Genomic instability and cancer: lessons from analysis of Bloom's syndrome. Biochemical Society Transactions, 37(3), 553-559.
5. Opresko, P. L., Calvo, J. P., & von Kobbe, C. (2007). Role for the Werner syndrome protein in the promotion of tumor cell growth. Mechanisms of ageing and development, 128(7), 423-436.
6. Pichierri, P., Ammazzalorso, F., Bignami, M., & Franchitto, A. (2011). The Werner syndrome protein: linking the replication checkpoint response to genome stability. Aging (Albany NY), 3(3), 311.
7. Wirtenberger, M., Frank, B., Hemminki, K., Klaes, R., Schmutzler, R. K., Wappenschmidt, B., ... & Burwinkel, B. (2006). Interaction of Werner and Bloom syndrome genes with p53 in familial breast cancer. Carcinogenesis,27(8), 1655-1660.
8. Hickson, I. D. (2003). RecQ helicases: caretakers of the genome. Nature Reviews Cancer, 3(3), 169-178.
9. Maxx Planck Institute of Biochemistry. http://www.biochem.mpg.de/en/rg/biertuempfel/research/index.html
10. Rossi, M. L., Ghosh, A. K., & Bohr, V. A. (2010). Roles of Werner syndrome protein in protection of genome integrity. DNA repair, 9(3), 331-344.
11. Ouyang, K. J., L. L. Woo, N. A. Ellis. 2008. Homologous recombination and maintenance of genome integrity: Cancer and aging through the prism of human RecQ helicases. Mech Aging Dev, 129: 425 - 440.