DNA Replication, Repair and Recombination 1 (Lec 3) Flashcards
What is the error rate in DNA replication?
1 mistake in every 10^9 base pairs.
True or False?
Germ cells have to have low mutation rates to maintain the species.
true
Why do somatic cells need low mutation rates?
to avoid uncontrolled proliferation/cancer
What reaction does DNA polymerase catalyze?
DNA synthesis
(DNA)n residues + dNTP -> (DNA) n + 1 residues + P2O7 ^4-
How is the DNA template directed?
new chain is assembled in a preexisting DNA template that is complementary to the incoming bases
True or False?
DNA replication requires dATP, dGTP, dCTP and dTTP
true
DNA polymerase requires a primer with a free ___ to begin
3’ -OH
What is the direction of growth of DNA?
5’ to 3’
Explain how the replication fork is asymmetric
Both strands are simultaneously replicated, DNA polymerase can only synthesize DNA in the 5’ to 3’ direction, leading strand is synthesized continuously, lagging strand is synthesized in segments
Where does the first step of proofreading occur?
just before a new nucleotide is added: enzyme must tighten its “fingers” around the active site, which is easier if the correct base is in place
When does exonucelolytic proofreading occur?
takes place immediately after incorrect base is added
What does 3’ to 5’ exonuclease do during proofreading?
clips off unpaired residues at 3’ primer terminus
How does 5’ to 3’ replication allow efficient error correction?
high energy bond is cleaved providing the energy for polymerization
How is the lagging strand synthesized?
backstitching process: DNA primase synthesizes a new RNA primer, DNA polymerase adds to new RNA primer to start new okazaki fragment, DNA plymerase finishes fragment, old RNA primer erased and replaced by DNA, nick sealed by DNA ligase
What is the protein at the DNA replication fork that unwinds DNA?
helicase
note: hydrolizes ATP which causes conformational change that propels it like a rotary engine along single stranded DNA
What do single-stranded DNA binding proteins do?
bind tightly and cooperatively to exposed single-stranded DNA; help stabilize unwound DNA, prevent formation of hairpins, and keep DNA bases exposed
What is the function of the sliding clamp at the replication fork?
keeps DNA polymerase on DNA when moving; releases when double stranded DNA is encountered
What is the difference in the clamp on the leading strand compared to the lagging strand?
leading strand: clamp remains associated to DNA polymerase for long stretches
lagging strand: clamp loader stays close so it can assemble a new clamp at start of each new okazaki fragment
In mismatch repair, how are errors detected?
errors are detected based on distortion caused by mispairing
What is the process of mismatch repair?
Binding of mismatch proofreading proteins (MutL and MutS) scan for the nick and trigger degradation of nicked strand, the repair is finalized with new DNA synthesis
What is DNA Topoisomerase?
a reversible enzyme that breaks a phosphodiester bond to change superhelicity, thereby relieving supercoiling
What does Type I Topoisomerase do?
catalyzes the relaxation of supercoiled DNA
How does Type I Topoisomerase work?
creates transient single strand break in DNA which allows the DNA on either side of the nick to rotate freely relative to each other; uses the other phosphodiester bond as a swivel point
What does Type II Topoisomerase do?
makes a transient double-stranded break in the DNA
How does Type II Topoisomerase work?
it uses ATP to 1) break double stranded helix reversibly to create “gate” 2) causes second strand to pass through 3) reseals break and dissociates
note: prevents severe tangling problems that would arise during DNA replication
What is the DNA replication origin?
A-T rich regions where sequence attracts initiator proteins to pry open DNA
How is DNA replication in bacteria initiated?
initiator proteins bind to specific sites in origin, forming complex which attracts DNA helicase + helicase loader
In regards to DNA replication in bacteria, once helicase loader is finished loading, what happens?
helicase unwinds DNA so primase can make RNA primer on leading strand; remaining proteins assemble to create 2 replication forks with complexes moving in opposite direction with respect to the origin
In terms of the cell cycle, where does Eukaryotic DNA replication occur?
during S phase
Different regions of each chromosome are replicated in a reproducible order during S phase, depending on ____ ____
chromatin structure
ex: heterochramtin is late-replicating ( X chromosomes of females)
Why is chromatin structure important for mammalian DNA replication origins?
they can still function if moved to a different locus if placed where chromatin is uncondensed
example: distant DNA in beta globin is required for expression of the genes in the cluster
Histone proteins are synthesized mainly in what phase of the cell cycle?
S phase
As replication fork passes through chromatin, histone octamer breaks into what?
an H3-H4 tetramer - distributed randomly to daughter duplexes
2 H2A-H2B dimers which are released form the DNA
Reassembly of the histones requires what type of protein?
chaperones; directed to DNA with sliding clamp called PCNA
What does Telomerase do?
replicates chromosome ends; replenishes special sequence at end of chromosome by elongating the parental strand in 5’ to 3’ direction using an RNA template on the enzyme
note: special sequence is GGGTTA
After extension of parental strand by telomerase, what happens?
replication of lagging strand can be completed by DNA polymerase, using extension as template
What does the telomere replication mechanism ensure?
3’ end is longer, leaving a protruding single stand end that loops back and tucks into the repeat (T-loop)
How is telomere length regulated?
each chromosome end in a given cell contains variable number of telomere repeats depending on age; after many generations, daughter cells will have defective chromosomes and stop dividing (replicative senescence)
Human fibroblasts normally divide how many times before undergoing replicative senescence?
60
