Question

You are studying cells cultured from patient sample and determine that they contain a large number of blocked DNA replication forks compared to control cells. You hypothesize that the patient cells would display a reduced number of blocked replication forks if you treated with a drug that activates which of the following? give brief explanation to your choice

l. Pre-replication complexes

ll. Protein kinase transducers

lll. Checkpoint sensor proteins.

lV. CDKs (cyclin-dependent kinases)

Answer 1

Treating the cultured patient cells with a drug that activates checkpiont sensor proteins may help reduce the number of blocked replication fork.

Researchers working with DNA replication also research replication fork (RF) dynamics, which is a key structural element of DNA synthesis. Hydroxyurea (HU) is used in laboratory experiments based on the cell cycle to model DNA damage during replication (Cell cycle; S phase).

HU causes deoxynucleotide triphosphates (dNTPs) to deplete and subsequently RF to stall. The RF's Y-shaped structure, which is at the center of action during DNA replication, guides the unwinding and DNA template synthesis. During this process, a host of proteins help RF and ensure that replication errors or DNA strand breakages are corrected before replication and cell cycle proceed further.

Some of the RF proteins form part of the fork protection complex (FPC), which also stabilizes the RF during arrest caused by HU. In the stalled RF, single-stranded DNA (ssDNA) generates a signal that activates the intra-S phase checkpoint and prevents the cell cycle from moving into the G2 phase until replication is complete.Once the replication barrier has been removed, either by adapting to the drug or deleting HU from the device, the DNA synthesis of the stalled RFs resumes.

Although some replication origins can start in late S phase (Lopes et al. 2001), completion of replication following arrest generally depends on restart of the stalled forks (Branzei & Foiani 2007). This reliance on restarting RFs to complete whole genome synthesis raises several important questions. What signal alerts the cell that replication has paused and RF progression has stopped? How do RFs restart replication once conditions improve or the error is resolved? These questions are particularly important to human health because maintenance of genome stability during S phase helps defend against cancer (Bartkova et al. 2005).

Checkpoint resolution and fork recovery depend on the creation of some ssDNA, as well as DNA damage repair, to restart replication. In addition, DNA nucleases help resolve stalled structures and are regulated by S phase checkpoint effector kinases: Chk1 in metazoa; Rad53 in Saccharomyces cerevisiae; Cds1 in Schizosaccharomyces pombe (Figure 1). Loss of these kinases results in excessive DNA unwinding in the presence of HU, producing an extended amount of ssDNA that becomes coated with RPA (Feng et al. 2006; Lucca et al. 2004; MacDougall et al. 2007; Sogo et al. 2002) and likely reflects uncoupling of the MCM helicase from the RF. Furthermore, the absence of effector kinases causes DNA damage with extensive formation of DSBs, which are likely caused by unresolvable DNA structures (Figure 5; Bailis et al, 2008; Feng et al. 2006; Lindsay et al. 1998; Lucca et al. 2004; Marchetti et al. 2002; Sogo et al. 2002).