Question

1. Which checkpoint and phase of the cell cycle is the expression of BRCA1 and BRCA2 the greatest? What is the function of BRCA1 and why would it make sense that its expression is high at the checkpoint/phase of the cell cycle?

2. Outline the two parts of the eukaryotic cell cycle and describe the three main checkpoints in cell cycle regulation.

Answer 1

1 Answer:-

a, BRCA1 is a large protein with multiple functional domains and interacts with numerous proteins that are involved in many important biological processes/pathways. Mounting evidence indicates that BRCA1 is involved in all phases of the cell cycle and regulates orderly events during cell cycle progression. BRCA1 deficiency, consequently causes abnormalities in the S-phase checkpoint, the G₂/M checkpoint, the spindle checkpoint and centrosome duplication. The genetic instability caused by BRCA1 deficiency, however, also triggers cellular responses to DNA damage that blocks cell proliferation and induces apoptosis. Thus BRCA1 mutant cells cannot develop further into full-grown tumors unless this cellular defense is broken. Functional analysis of BRCA1 in cell cycle checkpoints, genome integrity, DNA damage response (DDR) and tumor evolution should benefit our understanding of the mechanisms underlying BRCA1 associated tumorigenesis, as well as the development of therapeutic approaches for lethal disease.

The role of BRCA1 in the G₁/S cell cycle checkpoint needs further scrutiny, as BRCA1 deficient cells exhibit an intact G₁/S cell cycle checkpoint. p53 activation has obscured the role of BRCA1 in this checkpoint and further mechanistic studies should overcome this barrier. Second, it has been shown that expression of many genes that are critical for the spindle checkpoint is down regulated in cells carrying a targeted disruption or RNAi mediated acute suppression of BRCA 1

BRCA1 and BRCA2 encode very large proteins , widely expressed in different tissues during the S and G2 phases, which localize to the cell nucleus. They bear little resemblance to one another or to proteins of known function. Orthologs are not found in the yeast, fly, or worm genomes. Thus, BRCA1 and BRCA2 are relative latecomers in evolution, belying their apparently fundamental role in mammalian cells, and hinting at specialized, possibly tissue-specific, functions. These unusual features have at once confounded analyses of the biological function of the BRCA proteins,

Both BRCA1 and BRCA2 work to preserve chromosome structure, yet the precise nature of their contribution has proven difficult to define, because both proteins have been implicated in a multitude of different processes including DNA repair and recombination, cell cycle control, and transcription. Second, the similarities between the phenotypes induced by disruption of BRCA1 or BRCA2 and their claimed cohabitation in certain macromolecular complexes has prompted speculation that they work together in common cellular pathways.

b, Detection of BRCA1 transcripts initially did not identify a cell cycle component to its regulation. However, a study looking specifically at the status of BRCA1 mRNA in G0 cells found that the transcript was greatly reduced.⁴ While expression was high in exponentially growing cells, withdrawal of growth factor from human mammary epithelial cells resulted in a disappearance of BRCA1 altogether. In addition, senescent cells also had dramatically reduced BRCA1 transcript. The lack of BRCA1 transcript in non-dividing cells led to the notion that this may be a component of the cell division machinery. At least in the case of senescence, recent reports of the ability of p53 to repress the transcription of BRCA1 may underlie this result. Another contributor to the regulation of BRCA1 mRNA expression is the pRb-E2F complex. The promoter of BRCA1 contains several E2F binding sites and pRb is able to repress transcription from the BRCA1 promoter in an E2F-dependent fashion.

Development of antibodies against the full-length BRCA1 protein further established its role in the cell division process.the shift of the BRCA1 protein band to a higher mobility upon the onset of DNA replication, or if cells had been arrested in S phase by such agents as hydroxyurea. This shift was apparently due to phosphorylation, as addition of phosphatase to extracts resulted in a collapse of the band to its normal state, and phosphorylation occurred predominantly on serine . The phosphorylation of BRCA1 continued throughout S and onto the G2/M phases, after which it was progressively dephosphorylated. Blockage of cell cycle progression also resulted in phosphorylation, either at the G1/S border or at G2/M by treatment with colchicine. Later, BRCA1 was found to possess a cdk2 phosphorylation site at serine This site was found to be efficiently phosphorylated in vitro by cdk2 complexed with either cyclin A or E. Kinases complexed with cyclin D have also been shown to phosphorylate BRCA1. Therefore, at least one of the kinases that force the hyperphosphorylation of BRCA1 at S phase is a major component of the cell cycle machinery. To date, there exist several other kinases that are capable of BRCA1 phosphorylation including casein kinase 2, DNA damage-responsive kinases such as ATM, ATR, hCds1 and the AKT kinase as stimulated by heregulin. Whether these kinases also play a role in cell cycle-mediated alteration of BRCA1 remains to be tested. Also, it remains to be seen if cdk2 is capable of phosphorylating BRCA1 as late as G2/M phase—a phase that retains high levels of phosphorylated BRCA1 yet has classically been thought to possess low levels of cdk2 kinase activity. One report suggests that BRCA1 is actually predominantly tyrosine phosphorylated at G2/M stages, so there may be other cell cycle-regulated kinases that are able to affect BRCA1 phosphorylation status at different stages of the cycle. Thus far, the only concrete effect of phosphorylation on BRCA1 to be shown has been a change in protein-protein interaction with such regulators of its transcriptional activity as CtIP. There exists correlative evidence suggesting that phosphorylation may affect subcellular localization and the conferring of sensitivity to DNA damage, but no data thus far have placed a functional significance on cell cycle-dependent phosphorylation of BRCA1.

2 Answer:-

A checkpoint is a stage in the eukaryotic cell cycle at which the cell examines internal and external cues and "decides" whether or not to move forward with division.

The three most important ones are:

• The G_11​start subscript, 1, end subscript checkpoint, at the G_11​start subscript, 1, end subscript/S transition.
• The G_22​start subscript, 2, end subscript checkpoint, at the G_22​start subscript, 2, end subscript/M transition.
• The spindle checkpoint, at the transition from metaphase to anaphase.

G1 checkpoint is near the end of G1 (close to the G1/S transition). G2 checkpoint is near the end of G2 (close to the G2/M transition). Spindle checkpoint is partway through M phase, and more specifically, at the metaphase/anaphase transition.

The G1 checkpoint is the main decision point for a cell – that is, the primary point at which it must choose whether or not to divide. Once the cell passes the G 1, end subscript checkpoint and enters S phase, it becomes irreversibly committed to division. That is, barring unexpected problems, such as DNA damage or replication errors, a cell that passes the G 1, end subscript checkpoint will continue the rest of the way through the cell cycle and produce two daughter cells.

At the G 1, end subscript checkpoint, a cell checks whether internal and external conditions are right for division. Here are some of the factors a cell might assess:

• Size. Is the cell large enough to divide

• Nutrients. Does the cell have enough energy reserves or available nutrients to divide

• Molecular signals. Is the cell receiving positive cues (such as growth factors) from neighbors

• DNA integrity. Is any of the DNA damaged

G2 checkpoint

o make sure that cell division goes smoothly (produces healthy daughter cells with complete, undamaged DNA), the cell has an additional checkpoint before M phase, called the G2, end subscript checkpoint. At this stage, the cell will check:

• DNA integrity. Is any of the DNA damaged

• DNA replication. Was the DNA completely copied during S phase

If errors or damage are detected, the cell will pause at the G 2checkpoint to allow for repairs. If the checkpoint mechanisms detect problems with the DNA, the cell cycle is halted, and the cell attempts to either complete DNA replication or repair the damaged DNA.

If the damage is irreparable, the cell may undergo apoptosis, or programmed cell death. This self-destruction mechanism ensures that damaged DNA is not passed on to daughter cells and is important in preventing cancer.

The spindle checkpoint

The M checkpoint is also known as the spindle checkpoint: here, the cell examines whether all the sister chromatids are correctly attached to the spindle microtubules. Because the separation of the sister chromatids during anaphase is an irreversible step, the cycle will not proceed until all the chromosomes are firmly attached to at least two spindle fibers from opposite poles of the cell.