We have investigated the DNA substrate specificity of BACH1 (BRCA1-associated C-terminal helicase). The importance of various DNA structural elements for efficient unwinding by purified recombinant BACH1 helicase was examined. The results indicated that BACH1 preferentially binds and unwinds a forked duplex substrate compared with a duplex flanked by only one single-stranded DNA (ssDNA) tail. In support of its DNA substrate preference, helicase sequestration studies revealed that BACH1 can be preferentially trapped by forked duplex molecules. BACH1 helicase requires a minimal 5 ssDNA tail of 15 nucleotides for unwinding of conventional duplex DNA substrates; however, the enzyme is able to catalytically release the third strand of the homologous recombination intermediate D-loop structure irrespective of DNA tail status. In contrast, BACH1 completely fails to unwind a synthetic Holliday junction structure. Moreover, BACH1 requires nucleic acid continuity in the 5 ssDNA tail of the forked duplex substrate within six nucleotides of the ssDNA-dsDNA junction to initiate efficiently DNA unwinding. These studies provide the first detailed information on the DNA substrate specificity of BACH1 helicase and provide insight to the types of DNA structures the enzyme is likely to act upon to perform its functions in DNA repair or recombination.Germ line mutations in BRCA1 lead to an increased lifetime risk of breast and/or ovarian cancer. Cellular studies have revealed that the BRCA1 tumor suppressor gene is required for the maintenance of genomic integrity and a normal level of resistance to DNA damage (for review see Refs. 1-3). The nuclear phosphoprotein BRCA1 contains tandem C-terminal BRCT motifs, a conserved protein sequence found in a large number of DNA damage-response proteins (4). The integrity of the BRCT motifs is required for the role of BRCA1 in double strand break repair (DSBR) 1 and homologous recombination (5-8). Tumor-predisposing missense and deletion mutations in the BRCA1 BRCT domain, all of which render BRCA1 defective in its DSBR function, also disrupt the ability of BRCA to bind BACH1. BACH1 is a member of the DEAH subfamily of superfamily 2 helicases (9). Consistent with its predicted helicase domain, BACH1 was recently shown to catalyze DNA unwinding of M13 partial duplex substrates and have a 5Ј to 3Ј polarity on a linearized M13 directionality substrate (10). A role of BACH1 helicase in DSBR was suggested by the observation that overexpression of a BACH1 allele (K52R) carrying a mutation in its ATP-binding pocket that inactivates its ATPase/ helicase function (10) resulted in a marked decrease in the ability of cells to repair DSBs, and that this dominant negative phenotype depended on a specific interaction between BACH1 and BRCA1 (9). More recently, it was shown that the interaction between BRCA1 and BACH1 depends on the phosphorylation status of BACH1 (11-13), and that this phosphorylationdependent interaction is required for DNA damage-induced checkpoint control during the G 2 /M phase of the ...

No abstract

Mutations in the FANCJ helicase predispose individuals to breast cancer and are genetically linked to the Fanconi anemia (FA) complementation group J. FA is a chromosomal instability disorder characterized by multiple congenital anomalies, progressive bone marrow failure, and high cancer risk. FANCJ has been proposed to function downstream of FANCD2 monoubiquitination, a critical event in the FA pathway. Evidence supports a role for FANCJ in a homologous recombination pathway of double strand break repair. In an effort to understand the molecular functions of FANCJ, we have investigated the ability of purified FANCJ recombinant protein to use its motor ATPase function for activities in addition to unwinding of conventional duplex DNA substrates. These efforts have led to the discovery that FANCJ ATP hydrolysis can be used to destabilize protein-DNA complexes and unwind triple helix alternate DNA structures. These novel catalytic functions of FANCJ may be important for its role in cellular DNA repair, recombination, or resolving DNA structural obstacles to replication. Consistent with this, we show that FANCJ can inhibit RAD51 strand exchange, an activity that is likely to be important for its role in controlling DNA repair through homologous recombination.A growing interest in the mechanisms of helicases in cellular nucleic acid metabolism has been partly fueled by the knowledge that an increasing number of human diseases are genetically linked to mutations in genes encoding helicase-like proteins. RecQ helicases are prominent in this category, since three diseases of premature aging and/or cancer are attributed to recessive mutations in genes that encode bona fide DNA helicases (Werner and Bloom Syndromes) or DNA-dependent ATPase (Rothmund-Thomson Syndrome) (1, 2). Other helicase disorders exist, including Fanconi anemia (FA), 3 which is a recessive genetic disorder characterized by multiple congenital anomalies, progressive bone marrow failure, and high risk of cancer (for a review, see Ref.3). Among the 13 FA complementation groups from which all of the FA genes have been cloned, the FANCM and FANCJ genes encode DNA-stimulated ATPases, the latter also being a Superfamily 2 DNA helicase (4). The interaction of FANCJ with BRCA1 (5) and the existence of FANCJ mutations in early onset breast cancer patients (5, 6) as well as its genetic linkage to the FA-J complementation group (7-9) have clarified that FANCJ is a tumor suppressor.Although FANCJ helicase activity has been studied biochemically, the cellular functions of FANCJ in DNA repair are likely to require additional functions that involve protein-protein and protein-DNA interactions. Two protein partners of FANCJ that appear to be important are the single-stranded DNA-binding protein RPA, which stimulates FANCJ helicase activity (10), and the mismatch repair complex MutL␣, to which FANCJ binds and enables FANCJ to perform its DNA repair function (11). The FANCJ helicase functions downstream of FANCD2 monoubiquitination, a critical event in the FA pathway (9...

Infarction occurs when myocardial perfusion is interrupted for prolonged periods of time. Short episodes of ischemia and reperfusion protect against tissue injury when the heart is subjected to a subsequent prolonged ischemic episode, a phenomenon known as ischemic preconditioning (IPC). Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that mediates adaptive responses to hypoxia/ischemia and is required for IPC. In this study, we performed a cellular and molecular characterization of the role of HIF-1 in IPC. We analyzed mice with knockout of HIF-1α or HIF-1β in Tie2 + lineage cells, which include bone marrow (BM) and vascular endothelial cells, compared with control littermates. Hearts were subjected to 30 min of ischemia and 120 min of reperfusion, either as ex vivo Langendorff preparations or by in situ occlusion of the left anterior descending artery. The IPC stimulus consisted of two cycles of 5-min ischemia and 5-min reperfusion. Mice lacking HIF-1α or HIF-1β in Tie2 + lineage cells showed complete absence of protection induced by IPC, whereas significant protection was induced by adenosine infusion. Treatment of mice with a HIF-1 inhibitor (digoxin or acriflavine) 4 h before Langendorff perfusion resulted in loss of IPC, as did administration of acriflavine directly into the perfusate immediately before IPC. We conclude that HIF-1 activity in endothelial cells is required for acute IPC. Expression and dimerization of the HIF-1α and HIF-1β subunits is required, suggesting that the heterodimer is functioning as a transcriptional activator, despite the acute nature of the response.T he heart requires a constant supply of O 2 for generation of ATP, 95% of which is derived from oxidative phosphorylation (1). Coronary artery stenosis due to an atherosclerotic plaque results in reduced perfusion and myocardial ischemia, especially under conditions of increased myocardial O 2 demand, as occurs when heart work is increased in response to physical exertion or emotional stress. Plaque rupture is a catastrophic event that results in complete arterial occlusion and, within ∼20

The BRCA1 associated C-terminal helicase (BACH1) associated with breast cancer has been implicated in double strand break (DSB) repair. More recently, BACH1 (FANCJ) has been genetically linked to the chromosomal instability disorder Fanconi Anemia (FA). Understanding the roles of BACH1 in cellular DNA metabolism and how BACH1 dysfunction leads to tumorigenesis requires a comprehensive investigation of its catalytic mechanism and molecular functions in DNA repair. In this study, we have determined that BACH1 helicase contacts with both the translocating and the non-translocating strands of the duplex are critical for its ability to track along the sugar phosphate backbone and unwind dsDNA. An increased motor ATPase of a BACH1 helicase domain variant (M299I) enabled the helicase to unwind the backbone-modified DNA substrate in a more proficient manner. Alternatively, increasing the length of the 5′ tail of the DNA substrate allowed BACH1 to overcome the backbone discontinuity, suggesting that BACH1 loading mechanism is critical for its ability to unwind damaged DNA molecules.

Promising research on DNA repair signaling pathways predicts a new age of anti-tumor drugs. This research was initiated through the discovery and characterization of proteins that functioned together in signaling pathways to sense, respond, and repair DNA damage. It was realized that tumor cells often lacked distinct DNA repair pathways, but simultaneously relied heavily on compensating pathways. More recently, researchers have begun to manipulate these compensating pathways to reign in and kill tumor cells. In a striking example it was shown that tumors derived from mutations in the DNA repair genes, of BRCA-FA pathway, were selectively sensitive to inhibition of the base excision repair pathway. These findings suggest that tumors derived from defects in DNA repair genes will be easier to treat clinically, providing a streamlined and targeted therapy that spares healthy cells. In the future, identifying patients with susceptible tumors and discovering additional DNA repair targets amenable to anti-tumor drugs will have a major impact on the course of cancer treatment. KeywordsFanconi anemia; DNA repair; anti-tumor drug; BRCA The DNA damage responseThe integrity of the genome is under constant attack from DNA damage. Even internal processes such as normal cellular metabolism create byproducts that wreak havoc on DNA. In some instances, DNA damage is severe enough to create a double strand break in the DNA helix. If breaks are not repaired, genes can be extensively rearranged and chromosomes can become unstable. To avoid this type of genomic instability, cells monitor DNA for signs of damage and respond by mounting a DNA damage response. The response is initiated by distortions in the DNA structure and eventually leads to activation of both cell cycle checkpoints and DNA damage repair pathways.Checkpoints can be triggered by DNA damage at several stages in the cell cycle, e.g., the G1 to S-phase and the G2 to M-phase boundaries. DNA damage can also arrest the replication of DNA within S phase, when new DNA is synthesized. Arresting DNA synthesis provides cells sufficient time to repair damaged DNA before progressing into G2 and M phases. Thus, cell cycle checkpoints cooperate with DNA repair processes to ensure that the genome replicates accurately. If, however, the damage is irreparable, the cell will activate an apoptotic response

It has been proposed that selective inactivation of a DNA repair pathway may enhance anti-cancer therapies that eliminate cancerous cells through the cytotoxic effects of DNA damaging agents or radiation. Given the unique and critically important roles of DNA helicases in the DNA damage response, DNA repair, and maintenance of genomic stability, a number of strategies currently being explored or in use to combat cancer may be either mediated or enhanced through the modulation of helicase function. The focus of this review will be to examine the roles of helicases in DNA repair that might be suitably targeted by cancer therapeutic approaches. Treatment of cancers with anti-cancer drugs such as small molecule compounds that modulate helicase expression or function is a viable approach to selectively kill cancer cells through the inactivation of helicase-dependent DNA repair pathways, particularly those associated with DNA recombination, replication restart, and cell cycle checkpoint.

The genetic complexity of cancer has posed a formidable challenge to devising successful therapeutic treatments. Tumor resistance to cytotoxic chemotherapy drugs and radiation which induce DNA damage has limited their effectiveness. Targeting the DNA damage response is a strategy for combating cancer. The prospect for success of chemotherapy treatment may be improved by the selective inactivation of a DNA repair pathway. A key class of proteins involved in various DNA repair pathways is comprised of energy-driven nucleic acid unwinding enzymes known as helicases. DNA helicases have been either implicated or have proposed roles in nucleotide excision repair, mismatch repair, base excision repair, double strand break repair, and most recently cross-link repair. In addition to DNA repair, helicases have been implicated in the cellular processes of replication, recombination, transcription, and RNA stability/processing. The emerging evidence indicates that helicases have vital roles in pathways necessary for the maintenance of genomic stability. In support of this, a growing number of human genetic disorders are attributed to mutations in helicase genes. Because of their essential roles in nucleic acid metabolism, and more specifically the DNA damage response, helicases may be a suitable target of chemotherapy. In this review, we have explored this hypothesis and provided a conceptual framework for combinatorial treatments that might be used for combating cancer by inhibiting helicase function in tumor cells that already have compromised DNA repair and/or DNA damage signaling. This review is focused on helicase pathways, with a special emphasis on DNA cross-link repair and double strand break repair, that impact cancer biology and how cancer cells may be chemosensitized through the impairment of helicase function.