Sjogren: Lecture 3 DNA replication: regulating initiation and connection to disease Flashcards
What is important to know about replication?
Initiated at numerous origin sites (ori).
- ori spaced about 10^5 bp apart
DNA melting is a key regulatory step to initiate replication.
Runs bidirectionally and need topoisomerase 2 for termination and segregation of chromosomes.
What are the steps of replication initiation?
During G1, licensing factors - CTD1 and CDC6 are recruited to ORC to form preinitiation complex.
- 1 licensing/cycle
Activation of replication occurs in S phase
- Cell cycle kinases (CDK2, DDK) phosphorylate the licensing factors (Cdc6, Cdt1 and MCM).
Phosphorylated licensing factors Cdc6 and Cdt1 are released. MCM helicase is activated.
What is the order of replication firing and elongation?
After the licensing factors are phosphorylated, the addition Cdc45+GINS opens to ssDNA and formation of 2 diverging replication forks.
ssDNA stabilized by replication protein A (RPA).
What is important to know about PCNA
This sliding clamp called Proliferating Cell Nuclear Antigen (PCNA) bunds with the clamp loader; Replication Factor C (RFC).
DNA will bind to the RFC/PCNA complex and convert into processive enzymes.
Replication will occur until replicons meet fuse.
Explain licensing factors. What they do, how they do it and why they are important?
Licensing factors CTD 1 and CDC6 are recruited to the origin of replication complex (ORC) to help form preinitiation complex.
This is important for limited DNA replication to once per cell cycle.
CDK2 and DDK are cell cycle kinases that phosphorylate the licensing factors MCM, Cdc6 and Ctd1.
Phosphorylated forms of MCM, Cdc6 and Ctd1 can no longer license the start of a second round DNA replication.
How do cyclins/CDK regulate replication?
Cyclin dependent kinases (CDKs) are activated by binding to cyclins.
The expression of cyclins is tightly controlled in sync with the cell cycle.
Important to note the interaction of Cyclin A and CDK2.
Importance of Cyclin A?
Transcribed at onset of DNA synthesis.
Transcription is mediated by cAMP and mitogen growth factors.
Involves TF E2F and loss of repressing gene transcription.
Indirectly regulated by tumor suppressor protein p53.
How does the dysfunction of DNA replication and repair lead to cancer?
Cancer is caused by changes in the genome that lead to unregulated excessive division of cells.
Dysfunction of DNA repair causes cancer.
What are some anticancer agents that target DNA replication and respond to DNA damage?
Most successful approaches inhibit biosynthesis of the DNA building blocks: dATP, dGTP, dCTP, dTTP.
One target of synthesis of dTMP from dUMP by thymidylate synthase (TS)
-Direct inhibition using 5-FU
-Indirect inhibition using Methotrexate (MTX)
What is the basis for HIV drug action and selectivity?
AZT and ZDV are selective for Reverse Transcriptase which is present in the virus and not humans so the inhibition can only occur in the virus.
You can’t inhibit what isn’t present.
What is the MOA and significance of alkylating agents?
Alkylating agents cause a cross-link between DNA strands using the Guanine N7 position. This breaks the DNA, inhibits replication and lead to cell arrest - inhibit proliferation.
How can recombination events lead to cancer?
Philadelphia chromosome and BCR-ABL can have an unequal exchange during recombination of chromosome 9 and 22.
The ABL (tyrosine kinases) gene region with auto-inhibition is lost and this kinase remains active at all times.
-This will send signals for cell proliferation even is no stimulatory cytokine factor is present.
found in nearly all cases of chronic myelogenous leukemia (CML)
