DNA Replication- Lecture 29 Flashcards
DNA replication begins at ______ which are located on average _____.
Origins of replication (oris)
100,000 base pairs apart
_____ is the term used to describe initiation of DNA replication from an origin.
Origin firing
Describe the pathway of bi-directional replication.
DNA polymerase synthesizes DNA using each strand as a template starting at an ori and the “replication fork” representing newly synthesized DNA moves away from the ori in both directions simultaneously without releasing for hundreds of thousands of nucleotides
What are the two parts of the newly replicated DNA?
the leading and lagging (Okazaki fragments) strands
What are the DNA polymerases involved in DNA replication?
alpha- primer synthesis (DNA primase activity)
beta- base excision repair
gamma- mitochondiral DNA polymerase
delta- major lagging strand synthesis and MMR function
epsilon- major leading strand synthesis and NER functions
zeta- inaccurate replication at thyamine dimer
eta- adds As opposite thyamine dimers
What are the four phases of the cell cycle?
Gap phase (G1) S phase (S) Gap phase (G2) Mitosis (M)
What is the first step of DNA replication?
formation of the pre-initiation complex- binding at an ori by the Origin Recognition Complex (ORC) which recruits additional proteins (topoisomerase and single stranded DNA binding protein) and the replication machinery
Unlike synthesis by RNA polymerase, DNA synthesis proceeds only from a _______ that provides a free ____ on to which DNA polymerase adds the next nucleotide.
pre-existing “primer”
3’ hydroxyl
Describe the pathway of initiation?
DNA primase subunit of DNA polymerase alpha) lays down a short RNA primer, which is then extended by DNA polymerase (once for the leading strand and many times for the lagging) –> RNA primers are eventually replaced with DNA and all DNA fragments are ligated together to forma continuous double-helix from one end of the chromosome to the other
How are RNA primers removed from Okazaki fragments?
“old” RNA primer (located ahead of a new primer extension product) is displaced by DNA polymerase –> “flap structure with the RNA no longer annealed to the DNA template strand –> RNA primer is cleaved off endonucleolytically by FEN1 (a flap endonuclease) –> nick between the 5’ end of the “old” Okazaki DNA and the 3’ end of the “new” Okazaki DNA is sealed by DNA ligase
_____ are commonly used as antivirals.
Nucleoside analogues that do not have a 3’ hydroxyl onto which a subsequent nucleotide can be added
The ends of chromosomes are called _____.
telomeres
What is the repeat sequence for telomeric DNA?
TTAGGG
How is the telomeric region distinguished from a broken DNA end?
the folding of the DNA back into a structure called a t-loop
Why are t-loops important?
prevents end-to-end joining of chromosomes and signal that an end is not broken (which indicates catastrophic DNA damage that can result in apoptosis)
How are telomere repeat sequences added?
by telomere-specific proteins that form the “shelterin” complex
What is the end replication problem?
the nature of the lagging strand does not allow for the very end to be fully synthesized, resulting in the telomere shortening with each round of replication
How do cells solved the end replication problem?
a reverse transcriptase known as “telomerase” (a ribonucleoprotein complex that carries its own RNA template and that can extend the lagging strand)
Why do only some cells have telomerase activity?
because normal somatic cells do not need to continually replicate, so they are limited to a number of cell divisions before they become senscent (the Hayflick limit)
In some cancers, the reactivation of _____ allows cells to continue to divide unchecked.
telomerase
What is the mechanism of telomerase based DNA extension?
telomerase RNA binds to a complementary sequence in the lagging strand template –> lagging strand template is extended by six nucleotides based on the sequence of the RNA template carried by telomerase –> telomerase binds again at its complementary sequence (six nucleotides downstream of the last one) and the reaction is repeated
