LINK BETWEEN CELL CYCLE AND DNA REPLICATION

-BY SHREYA AHUJAROLL NO. 11

REPLICATION AND CELL CYCLEWhether a cell has only one chromosome (as in prokaryotes) or many chromosomes (as in eukaryotes), the entire genome must be replicated precisely once for every cell division.

How is this act of replication linked to cell cycle?

Two general principles are used to compare the state of replication with the condition of the cell cycle:

• Initiation of DNA replication commits the cell (prokaryote or eukaryote) to a further division. Replication is controlled at the stage of initiation. Once replication has started, it continues until the entire genome has been duplicated.

• If replication proceeds, the consequent division cannot be permitted to occur until the replication event has been completed. Indeed, the completion of replication may provide a trigger for cell division. The duplicate genomes are then segregated one to each daughter cell.

Trigger for cell division

• In prokaryotes, the initiation of replication is a single event involving a unique site on the bacterial chromosome, and the process of division is accomplished by the development of a septum that grows from the cell wall and divides the cell into two.

• In eukaryotic cells, initiation of replication is identified by the start of S-Phase, , a protracted period during which DNA synthesis occurs, and which involves many individual initiation events. The act of division is accomplished by the reorganization of the cell at mitosis.

• The unit of DNA in which an individual act of replication occurs is called the replicon. Each replicon “fires” once and only once in each cell cycle. It has an origin at which replication is initiated and a terminus at which replication stops.

• Each eukaryotic chromosome contains a large number of replicons; thus the unit of segregation includes many units of replication. This adds another dimension to the problem of control: All of the replicons must be fired during one cell cycle. They are not, however, active simultaneously. But each replicon must be activated no more than once in each cell cycle.

• Hence, some signals must distinguish the replicated from the non-replicated replicons to ensure that replicons do not fire a second time. And since, many replicons are activated independently, there must be another signal to indicate when the entire process of replicating all replicons is completed.

• Additionally, there are multiple copies of each organelle DNA per cell, and the control of organelle DNA replication must be related to cell cycle.

FACTS ABOUT INITIATION OF REPLICATION

AUTONOMOUSLY REPLICATING SEQUENCE IN EUKARYOTES• An autonomously replicating sequence (ARS) contains the origin of replication in

the yeast genome. It contains four regions (A, B1, B2, and B3), named in order of their effect on plasmid stability; when these regions are mutated, replication does not initiate.

• As seen above the ARS are considerably A-T rich which makes it easy for replicative proteins to disrupt the H-bonding in that area. ORC protein complex (Origin Recognition Complex) is bound at the ARS throughout the cell cycle, allowing replicative proteins access to the ARS.

• Mutational analysis for the yeast ARS elements have shown that any mutation in the B1, B2 and B3 regions result in a reduction of function of the ARS element. A mutation in the A region results in a complete loss of function.

• Melting of DNA occurs within domain B2, induced by attachment of ARS Binding factor 1 to B3. A1 and B1 domain binds with Origin Recognition Complex.

• 5'- T/A T T T A Y R T T T T/A -3

• The process of duplicating DNA is called DNA replication, and it takes place by first unwinding the duplex DNA molecule, starting at many locations called DNA replication origins, followed by an unzipping process that unwinds the DNA as it is being copied.

• However, replication does not start at all the different origins at once. Rather, there is a defined temporal order in which these origins fire. Frequently a few adjacent origins open up to duplicate a segment of a chromosome, followed some time later by another group of origins opening up in an adjacent segment.

• Replication does not necessarily start at exactly the same origin sites every time, but the segments appear to replicate in the same temporal sequence regardless of exactly where the segments are.

• PRIMING OF DNA REPLICATION: DNA double helix is unwound and an initial priming event by DNA polymerase α occurs on the leading strand. Priming of the DNA helix consists of synthesis of an RNA primer to allow DNA synthesis by DNA polymerase α. Priming occurs once at the origin on the leading strand and at the start of each Okazaki fragment on the lagging strand.

• DNA replication is initiated from specific sequences called origins of replication, and eukaryotic cells have multiple replication origins. To initiate DNA replication, multiple replicative proteins assemble on and dissociate from these replicative origins. The individual factors described below work together to direct the formation of the pre-replication complex (pre-RC), a key intermediate in the replication initiation process.

PRE-REPLICATIVE COMPLEX

REGULATION OF DNA REPLICATION

ACTIVATION OF CDK

Y - Tyrosine residuesT – Threonine residuesCAK – CDK activating kinase

o CDK is the key regulator of pre-RC assembly

o CDK acts by inhibiting the individual components of the pre-RC. CDK phosphorylates Cdc6 to mark it for degradation by the SCF in late G1 and early S phase

o CDK also phosphorylates ORC proteins. It has been suggested that phosphorylation affects the ability of the ORC to bind other components of the pre-RC.

o In metazoans, studies have shown that Geminin prevents pre-RC assembly by binding to cdt1 and preventing its association with the pre-RC. Since geminin is degraded by the APC/C, pre-RC assembly can proceed only when APC/C activity is high, which occurs in G1.

Origin licensing: The hexameric replicative helicase, Mcm2-7, is loaded in an inactive form into pre-replicative complexes (pre-RCs) at potential origin sites. This helicase loading step requires the ATP-dependent activity of the Origin Recognition Complex (ORC), which marks potential origin sites on the chromosome, Cdc6, which like several of the ORC subunits is a member of the AAA+ family of ATPases.

ORC and Cdc6 cooperatively load two heptamers of the Cdt1·Mcm2-7 complex into a head-to-head Mcm2-7 double hexamer onto DNA

Chromosomal sites containing functional pre-RCs serve as binding sites for additional replication factors including Mcm10, Sld3, and Cdc45. Function unclear.

DDK and CDK promote the activation of a subset of “licensed” origins by activating the MCM2-7 helicase and by promoting the assembly of further initiation factors around pre-RCs to form pre-initiation complexes or replisomes.

CDK phosphorylates two replication proteins, Sld3 and Sld2, both of which bind to Dpb11 when phosphorylated. Dpb11 has two pairs of tandem BRCT domains, known as a phosphopeptide-binding domain. The N-terminal pair of the BRCT domains binds to phosphorylated Sld3, and the C-terminal pair binds to phosphorylated Sld2. These phosphorylation-dependent interactions are essential, and represent the minimal requirement for CDK-dependent activation of DNA replication in budding. DDK phosphorylates Mcm, and this phosphorylation is thought to enhance the interaction between Mcm and other replication proteins by alleviating an inhibitory activity in Mcm4

CELL CYCLE IS HALTED IN RESPONSE TO DNA DAMAGE

Maintenance of the G1/S DNA damage checkpoint in the presence and absence of Cdk2. In response to DNA damage, activation of p53-p21 pathway is not altered in the absence of Cdk2, the primary target of p21 at the G1/S checkpoint. In the presence of Cdk2, the induced p21 inhibits Cdk2/cyclin E complexes in response to DNA damage. In the absence of Cdk2, Cdk1/cyclin E complexes are responsible for promotion of the G1/S transition and as a result become the target for p21 inhibition in response to DNA damage. Nevertheless, Cdk1 is not fully capable to rescue the functions of Cdk2 in DNA damage repair.