10: Replication fidelity Eukaryotic DNA replication Telomeres and telomerase

lec 10 and recap

what is good about anti mutators

has high quality for a slower time

• takes long but has high fidelity

what is mutogenesis used for

to induce random mutoations into a population of cells

what are cells with higher than normal mutation rated called

the mutated strains

that are cells with lower mutation rates

anti mutator strains

what is the first dna proof reading step carried out by

dna polymerase and occurs before the new nucleotide is added to the growing chain

• correct nucleotide fit with the complementary base has higher affinity for the moving polymerase compared to the incorrect nucleotide

• active site of polymerase only accommodates for the right size pairs

• after nucleotide binding ( before formation of a covalent bond) the polymerase undergo a conformational change ( closing fingers) and incorrectly pound nucleotides can be rejected

what is a con of ultra high fidetlity

thates a significant amount of time

• causes the trade off of speed or accuracey in the mutated strain an dthe antimutated strain

what helps direct/ coordinate phosphodiester formation

tyrosine and its rings, positive tyrosine and negative phosphates therfreo bind

what is the second proof reading step

• mismatch repair

• mediated 3’-5’ exonuclease activity of polymerase

  • actively removes misincorporated nucleotides

• if the wrong is added and continues that is a mismatch and can be detected and repaired

• when it is not the right since it stops, goes back. chews off with exo nuclease on that last nucleotide

• it is methyl directed and used to mark new form old and determine directionally the repair correctly

what is the final proofreading step

strand detection of mismatch repair mechanisms to further lower error rates in dna replication

what is mutS for

detects distortion of the dna helix and binds to a mismatched base pair

what is mutL for

binds the mutS-dna complex and activates mutH at near by methylated GATC to introduce a nick on the unmethylated strand of the dna duplex

once bound what does mutS and mutL do

can nearby dna for a nick, once detected mutl recruits helicase to separate dna strands and exonuclease to degrade the nicked strand all the way back pass the mismatch

what does dna pol 3 do during mismatch repair

comes in and fills in the gap using methylated strand as template

which strand will be methylated after replication

only the parental strand

what does mut h do

Activated by MuteL and detects the presence of a methylated nucleotide

• scans for methylated gatc on either side of a mismatch

why was molecular assy developed/ what does it involve

involves

• creating a double stranded plasmid with a mismatch

  • plasmid contains restriction enzyme recognition sites engineered for resistance

• incubation of cells

  • repaired plasmid is tested for the restriction enzyme sites

    • if restored the plasmid will be sensitive to the restriction enzyme confirming that the mismatch has been repaired

what phase does dna replication occur in

DNA replication only occurs during the S (synthesis) phase of the cell cycle.

difference between eukaryotic and prokaryotes in relation to genomes

eukaryotic cells have a much larger genome and are chromatized, linear chromosomes with many origins for replication

what are the cell cycle phases

G0, G1, S, G2, and M S phase

which has a slower rate of replication

eukaryotes are slower likely because of chromatin

what is ARS

autonomously replication sequences are origins in yeast that are well defined

how are origins found in eukaryotes

licensing factors such as origin recognition complex,

• they mark potential origins during g1 and later additional proteins, mcm2-7 complex and helicase are recruited to initiate replication

what do mcm2-7 proteins do

provide helicase activity for dna synthesis and loading of these proteins confers on the origin to fire in s phase

what is the protein needed for dna synthesis

the action of 2 protein kinases

why is protein kinases needed for dna synthesis

triggers the association of additional proteins with the origin

• during the process of initiation dna polymerase are also recruited at the start

where do mcm2-7 proteins go during replication

they move away from the origin and the pre replicative complexes get blocked

• this ensures that origins can only fire a single time per cell cycle

what does pol a do

primase + another dna polymerase activiy, it synthesizes both rna and dna primers

what synthesizes the leading strand

pol e

what synthesizes the lagging in strand

alternates between pol a and delta

how is high fidelity mainteined

base pairing, primer selection, proofreading

why does chromatin cause problems/ slow it down

chromatin needs to be disassembles ahead of the replication for and reasspemple nucleosomes post dna replication

• pcna directly binds to chormatin remodeling complexes such as caf 1

then nucleosomes are redistributed to new strands

which pol have high fidelity

alpha delta, epsilon

what is the end replication problem

. Every time DNA replicates, the very end of linear chromosomes cannot be replicated due to the absence of a primer for DNA synthesis.

what protective structure helps agains the end replication problem

telomeres

what are telomeres

• consist of repeating sequences like TTAGGG in humans.

• They exist at the ends of chromosomes and include both double-stranded and single-stranded regions.

• The single-stranded overhang is G-rich and extends beyond the double-stranded portion.

why are telomeres important

they preserve chromosome integrity

what is telomere length linked to

preventing cell aging d preserving genetic material

what does firdelity refer to

the accuracy of copying dna to prevent accumulation of mutations during cell division

overall facts for prokaryotic dna replication

• occurs in cytoplasm

• single region of origin

• dnag makes rna primer

• pol3= main replicative polymerase

• rna primer removed by rnase h

• replicated naked dna

• okazaki fragments are 1000-2000 nts in length

• replicates at 750-1000 per second

overall facts for eukaryotic replication

• occurs in nucleus

• many origins of replication

• pola makes rna and dna primer

• pol delta synthesizes lagging strand pole synthesizes leading strand

• rna primer removed by rnase h1 and fen1

• replicates dna in chromatin context

• okazaki fragments are 100-200nts

• replicates at 50-100 nts per second