Lect. 3 - Replication, repair and recombination 1 Flashcards
mutation rate in human germ line
one nucleotide change per 10^8 nucleotides per generation
mutation rate in e.coli
one nucleotide change per 10^10 nucleotides per cell generation
What is a critical need for multicellular organisms?
HIGH FIDELITY REPLICATION; Germ cells have to have low mutation rates to maintain the species and somatic cells need low mutation rate to avoid uncontrolled proliferation/cancer
DNA polymerase
synthesizes DNA by catalyzing the following rxn: (DNA)n residues + dNTP –> (DNA)n+1 residues + P2O7
What does DNA polymerase require to begin?
a primer w/ a free 3’ -OH to begin
What direction can the DNA polymerase synthesize in?
5’ to 3’ direction
How often does DNA polymerase make a mistake?
1 out of every 10^9 nucleotides copied, thanks to proofreading.
What step is first 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
Exonucleolytic proofreading
DNA polymerase requires a perfectly paired 3’ terminus’ 3’ to 5’ exonucleuase clips off unpaired residues at 3’ primer terminus
When does exonucleolytic proofreading take place?
immediately after incorrect bases is added
Why 5’ to 3’?
Allows for efficient error correction; that direction is energetically favorable to remove a new chain and start over.
Lagging strand synthesis
replicated through backstitching process
what does DNA primase do?
synthesizes an 10nt long RNA primer to prime DNA synthesis b/c if DNA polymerase initiated it it would increase mutation rate
RNA primer
erased by RNAseH (recognizes RNA/DNA hybrids) and replaced w/ DNA;
What joins the ends after DNA replaces the RNA?
DNA ligase
DNA helicase
unwinds DNA; protein w/ 6 identical subunits that binds and hydrolyzes ATP; this confomational change that propels it like a rotary engine along single stranded DNA, passing it through a center hole.
Capable of prying apart the helix at rates of 1000 nucleotide pairs/sec
Single-stranded DNA binding proteins
bind tightly and cooperatively to exposed SS DNA; it is less energetically favorable so will want to try and make a double strand if possible (or bind to itself = hairpin)
what 3 things do the single-stranded DNA binding proteins do?
- help stabilize unwound DNA
- prevent formation of hairpins
- DNA bases remain exposed
Sliding clamp
keeps DNA polymerase on DNA when moving; releases when double stranded DNA is encountered (allows long stretches of DNA rep. to occur)
clamp loader
hydrolyzes ATP as it loads the clamp onto a primer-template junction
On the leading strand, what does the clamp do?
remains associated to DNA polymerase for long stretches
On the lagging strand, what does the clamp loader do?
stays close so it can assemble a new clamp at start of each new Okazaki fragment
Mismatch Repair
removes (almost all) errors missed by proofreading by detecting distortion caused by mispairing
Which strand is correct?
the methylated strand is correct in e.coli and in humans, depends on single strand breaks - present on lagging strand before Okazaki fragments are ligated, leading strand not known.
MutS
binds to mismatch
MutL
scans for the nick and triggers degradation nicked strand
DNA Topoisomerases
reversible enzyme that breaks a phosphodiester bond to change superhelicity, thereby relieving supercoiling
what makes up one turn in DNA?
every 10 bp replicated
Type 1 Topoisomerases
Catalyze the relaxation of supercoiled DNA, a thermodynamically favorable process.
How do Type 1 Topoisomberases work?
by creating a 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 is the resealing like in type I Isomerases?
rapid and doesn’t require any additional energy since energy is stored in the phosphotyrosine linkage
Type II Topoisomerase
make a transient double-stranded break in the DNA
What do type II topoisomerase use ATP to do?
- break one double-stranded helix reversibly to create “gate”
- causes second strand to pass through
- reseals break and dissociates
decatenate
2 interlocked DNA circles that can be separated by type II topoisomerase
Where/when are type II topoisomerases activated?
at sites on chromosomes where 2 double-stranded helices cross each other
Replication origins
A-T rich (only 2 H bonds) regions where sequence attracts initiator proteins to pry open DNA
What is the only point of control for E. coli?
initiation - that is why it’s highly regulated
Regulation of initiation in e. coli
proceeds only when sufficient nutrients are present; refractory period - delay until new strand is methylated
initiation of DNA replication in bacteria
initiator proteins bind to specific sites in ORI, forming complex which attracts DNA helicase + helicase loader
where is helicase places?
around a SS DNA exposed by assembly of complex
Function of helicase
unwinds DNA so primase can make RNA primer on leading strand; remaining proteins assemble to create 2 replication forks w/ complexes moving in opposite direction w/ respect to the ORI
In Eukaryotic DNA Replication, when does it occur and how long does it last?
occurs during DNA synthesis phase (S) which lasts about 8 hrs for mammalian cells (so will have a lot of different origins of replication)
in eukaryotic DNA replication, what replicates first?
regions of genome w/ less condensed chromatin replicate first
in yeast (proks), what are the 3 minimum requirements for sequence to be ORI?
- must have binding site or ORC (origin recognition complex)
- Must have an AT rich stretch for easy unwinding
- Must have binding site for proteins that help attract ORC
what are the helicase loading proteins involved in regulation of proks
Cdc6 and Cdt1
In S phase, what does activated Cdks lead to?
dissociation of helicase loading proteins, activation of helicase, unwinding of DNA, and loading of DNA polymerase, etc.
Prevention of assembly of new ORC
prevent assembly of new ORC until next M phase resets cycle; single chance to form in G1 when Cdk activity is low;
When is the second chance for pre-replicative complexes to be activated
can be activated and disassembled in S phase when Cdks activity is high
What is an important part for mammalian ORIs?
Chromatin Structure
if ORIs are moved to a different locus can they still function?
Yes, as long as they are placed where chromatin is uncondensed
replication requires not only DNA replication but what else?
synthesis and assembly of new proteins
when are histone proteins mainly synthesized?
S phase; amount made is highly regulated to meet requirements
As replication fork passes through chromatin, histone octamer breaks into what?
- and H3-H4 tetramer, distributed RANDOMLY to daughter duplexes
- 2 H2A-H2B dimers which are released from the DNA
What does reassembly require?
Histone chaperones
What is the make up of H2A/H2B?
they are half old and half new - they are added at random to complete complex
What is the sliding clamp called that directs the chaperones (chromatin assembly factors)?
PCNA
Patterns of histone modification can be inherited
*Some daughter nucleosomes contain only parental histones or only new ones but most are hybrids of old and new
What replicates the chromosome ends?
telomerase
Why is end replication a problem on the lagging strand?
because there is no place for RNA primer - not a problem for bacteria b/c they have circular genomes
What special sequence is at the end of each chromosome?
GGGTTA - repeated about 1000x
Telomerase
enzyme that replenishes these sequences by elongating parental strand in 5’ to 3’ direction using an RNA template on the enzyme
Telomerase Replication
after extension of parental strand by telomerase, replication of lagging strand can be completed by DNA polymerase, using extension as template
What does the telomere replication mechanism ensure?
that the 3’ end is longer, leaving a protruding SS end that loops back and tucks into the repeat
T loops
structures protect ends and distinguishes them from broken ones that need to be repaired
Shelterin
protective chromosome cap made up of proteins
Replicative senescence
After many generations, daughter cells will have defective chromosomes and stop dividing; in this way the cell’s lifetime is regulated to guard against cancer
What may be responsible for aging in animals?
replicative senescence
