Setting the stage for cohesion establishment by the replication fork

Comment on: Rudra S, et al. Cell Cycle 2012; 2114-2

Catalytic Strand Separation by RECQ1 Is Required for RPA-Mediated Response to Replication Stress

SummaryThree (BLM, WRN, and RECQ4) of the five human RecQ helicases are linked to genetic disorders characterized by genomic instability, cancer, and accelerated aging [1]. RECQ1, the first human RecQ helicase discovered [2–4] and the most abundant [5], was recently implicated in breast cancer [6, 7]. RECQ1 is an ATP-dependent DNA-unwinding enzyme (helicase) [8, 9] with roles in replication [10–12] and DNA repair [13–16]. RECQ1 is highly expressed in various tumors and cancer cell lines (for review, see [17]), and its suppression reduces cancer cell proliferation [14], suggesting a target for anti-cancer drugs. RECQ1’s assembly state plays a critical role in modulating its helicase, branch migration (BM), or strand annealing [18, 19]. The crystal structure of truncated RECQ1 [20, 21] resembles that of E. coli RecQ [22] with two RecA-like domains, a RecQ-specific zinc-binding domain and a winged-helix domain, the latter implicated in DNA strand separation and oligomer formation. In addition, a conserved aromatic loop (AL) is important for DNA unwinding by bacterial RecQ [23, 24] and truncated RECQ1 helicases [21]. To better understand the roles of RECQ1, two AL mutants (W227A and F231A) in full-length RECQ1 were characterized biochemically and genetically. The RECQ1 mutants were defective in helicase or BM but retained DNA binding, oligomerization, ATPase, and strand annealing. RECQ1-depleted HeLa cells expressing either AL mutant displayed reduced replication tract length, elevated dormant origin firing, and increased double-strand breaks that could be suppressed by exogenously expressed replication protein A (RPA). Thus, RECQ1 governs RPA’s availability in order to maintain normal replication dynamics, suppress DNA damage, and preserve genome homeostasis

DNA damage induced p53 downregulates Cdc20 by direct binding to its promoter causing chromatin remodeling

CDC20 is a critical molecule in the Spindle Assembly Checkpoint (SAC). It activates the Anaphase promoting complex and helps a dividing cell to proceed towards Anaphase. CDC20 is overexpressed in many tumor cells which cause chromosomal instability. There have been limited reports on the mechanism of SAC's response to genotoxic stress. We show that ectopically expressed p53 or DNA damage induced endogenous p53 can downregulate Cdc20 transcriptionally. We have identified a consensus p53-binding site on the Cdc20 promoter and have shown that it is being used by p53 to bind the promoter and bring about chromatin remodeling thereby repressing Cdc20. Additionally, p53 also downregulates Cdc20 promoter through CDE/CHR element, but in a p21 independent manner. This CDE/CHR element-mediated downregulation occurs only under p53 overexpressed condition but not in the context of DNA damage. The present results suggest that the two CCAAT elements in the Cdc20 promoter are not used by p53 to downregulate its activity, as reported earlier

LIMD1 is more frequently altered than RB1 in head and neck squamous cell carcinoma: clinical and prognostic implications

Introduction: To understand the role of two interacting proteins LIMD1 and pRB in development of head and neck squamous cell carcinoma (HNSCC), alterations of these genes were analyzed in 25 dysplastic head and neck lesions, 58 primary HNSCC samples and two HNSCC cell lines. Methods: Deletions of LIMD1 and RB1 were analyzed along with mutation and promoter methylation analysis of LIMD1. The genotyping of LIMD1 linked microsatellite marker, hmlimD1, was done to find out any risk allele. The mRNA expression of LIMD1 and RB1 were analyzed by Q-PCR. Immunohistochemical analysis of RB1 was performed. Alterations of these genes were correlated with different clinicopathological parameters. Results: High frequency [94% (78/83)] of LIMD1 alterations was observed in the samples studied. Compare to frequent deletion and methylation, mutation of LIMD1 was increased during tumor progression (P = 0.007). Six novel mutations in exon1 and one novel intron4/exon5 splice-junction mutation were detected in LIMD1 along with a susceptible hmlimD1 (CA)20 allele. Some of these mutations [42% (14/33)] produced non-functional proteins. RB1 deletion was infrequent (27%). Highly reduced mRNA expression of LIMD1 (25.1 ± 19.04) was seen than RB1 (3.8 ± 8.09), concordant to their molecular alterations. The pRB expression supported this data. Tumors with LIMD1 alterations in tobacco addicted patients without HPV infection showed poor prognosis. Co-alterations of these genes led the worse patients’ outcome. Conclusions: Our study suggests LIMD1 inactivation as primary event than inactivation of RB1 in HNSCC development

Study of the regulation of spindle assembly checkpoint gene cdc20 and its role in genomic instability in Human cancer

We have also investigated the transcriptional regulation of CDC20 in a normal dividing cell. Since it has been known that CDC20 is expressed within the cell in a cyclic manner with a peak in expression during pro-metaphase and the surge decreases by lateanaphase, we reasoned that there must be a repressive effect on CDC20 throughout the rest of the cell cycle to maintain its expression just at the basal level. To examine our hypothesis, we treated cells with HDAC inhibitor Trichostatin A and found release in promoter activity, indicating HDACs bind and repress the CDC20 promoter. WD40 domain containing proteins have been implicated to act as transcriptional repressors and CDC20 being one such protein we examined whether it could regulate its own expression. Interestingly, our studies revealed that CDC20 could repress its own transcription. Further, we also showed that HDAC2 and Histone 3 methylation at lysine 9 were involved in this CDC20 mediated regulation of its own VIII Summary IX promoter. Domain mapping of the CDC20 protein revealed that the WD40 domain was not sufficient for this control and that the whole protein is necessary for the transcriptional repression. The functional implication of this repression was further investigated in a time course experiment using synchronized cells which indicated that the repression on the CDC20 promoter by CDC20 itself was released during pro-metaphase when more of CDC20 protein was necessary within the cell to drive the cell from metaphase to anaphase. When the levels of CDC20 are low in the cell the recruitment of CDC20 to its transcription repression complex also increased, thereby implicating CDC20 to be one of the controls for the cyclic expression of the protein. This differential promoter occupancy depending on the cell cycle stage led us to investigate whether post translational modifications of CDC20 were responsible in the mechanism. Our studies revealed that ubiquitinated form of CDC20 was not able to bind to the CDC20 promoter whereas the deubiquitinated form was a strong repressor for the promoter. This finding was in agreement with the expression pattern and the promoter occupancy of CDC20, as revealed through our studies. During metaphase CDC20 is ubiquitinated and is expressed in high amounts in the cell. During this phase we find very little amount of CDC20 to be bound to its promoter. From anaphase, the CDC20 level decreases within the cell and it is no longer ubiquitinated and we find that the promoter occupancy of the protein increases remarkably. All these taken together indicate that CDC20 plays a vital role in its own transcription and is capable of modulating its own expression by differentially regulating its expression in a cell cycle dependent manner. The region of the CDC20 promoter responsible for binding CDC20 is still under investigation and it would be an interesting find, since, this is the first study implicating CDC20 in transcriptional regulation

Getting Ready for the Dance: FANCJ Irons Out DNA Wrinkles

Mounting evidence indicates that alternate DNA structures, which deviate from normal double helical DNA, form in vivo and influence cellular processes such as replication and transcription. However, our understanding of how the cellular machinery deals with unusual DNA structures such as G-quadruplexes (G4), triplexes, or hairpins is only beginning to emerge. New advances in the field implicate a direct role of the Fanconi Anemia Group J (FANCJ) helicase, which is linked to a hereditary chromosomal instability disorder and important for cancer suppression, in replication past unusual DNA obstacles. This work sets the stage for significant progress in dissecting the molecular mechanisms whereby replication perturbation by abnormal DNA structures leads to genomic instability. In this review, we focus on FANCJ and its role to enable efficient DNA replication when the fork encounters vastly abundant naturally occurring DNA obstacles, which may have implications for targeting rapidly dividing cancer cells

Getting Ready for the Dance: FANCJ Irons Out DNA Wrinkles

Mounting evidence indicates that alternate DNA structures, which deviate from normal double helical DNA, form in vivo and influence cellular processes such as replication and transcription. However, our understanding of how the cellular machinery deals with unusual DNA structures such as G-quadruplexes (G4), triplexes, or hairpins is only beginning to emerge. New advances in the field implicate a direct role of the Fanconi Anemia Group J (FANCJ) helicase, which is linked to a hereditary chromosomal instability disorder and important for cancer suppression, in replication past unusual DNA obstacles. This work sets the stage for significant progress in dissecting the molecular mechanisms whereby replication perturbation by abnormal DNA structures leads to genomic instability. In this review, we focus on FANCJ and its role to enable efficient DNA replication when the fork encounters vastly abundant naturally occurring DNA obstacles, which may have implications for targeting rapidly dividing cancer cells