DNA replication Flashcards
how are nucleotides joined to DNA sequences
nucleophilic attack (transfer of electron forming diester bond) on phosphate of new DNTP = binds new nucleotide and releases 2 phosphates
how does DNA replication take place
- DNA strands are unwound by DNA helicase
- DNA polymerase binds at 3’ ligases strands back together
what are Okazaki fragments
- RNA primers bind to DNA and act as template for DNA polymerase to bind to
- allow DNA polymerase to bind
where are okazaki fragments found
- lagging strand = 5’→3’
- needs okazaki fragments
- leading strand = 3’→5’
- DNA synthesised continually as DNA P binds to 3’ OH
why does DNA polymerase require primers
act as a hook to be able to bind to the lagging strand
function of dna primase
produces primers
binds to short RNA sequence and inserts DNA hybrids for DNA polymerase to bind to
what primers are produced on the leading strand
- short RNA primer is synthesised using template and NTPs by DNA Primase
- once RNA primer is in place, DNA polymerase extends it via 3’
what primers are produced on the lagging strand
- DNA primase makes RNA primer
- primase end dissociates and joins further up strand
- DNA polymerase extends RNA primer
- ribonuclease H removes RNA primer = leaving gap
- DNA polymerase extends across gap
- DNA ligase seals nick
diseases associated with replication errors
- mutation in genes encoding DNA helicase → werner syndrome (premmie aging)
- bloom gene mutation → bloom syndrome
- LOF in RecQ DNA helicase
what is speed of DNA polymerase dependent on
processivity (ability to catalyse consecutive reactions without losing the substrate)
what is a high processivity for an enzyme?
- 1 nuc/sec = non-processive
- multiple nuc/sec = processive
average no. nucleotides added each time enzyme binds to template
function of sliding clamp
-> increases processivity of dna polymerase
- dependent on ATP
- binds to primer:template junction via clamp loader
- ATP breaks off → clamp loader releases → DNA polymerase binds on
what are single stranded dna binding proteins (SSBs)
- hairpins when DNA coils up = stop polymerase
- SSBs bind to RNA and prevent hairpins
DNA topoisomerases function
- prevent DNA from being tangled during replication
- enhance processivity of DNA polymerase
- DNA topoisomerases relax tension resealing the backbone of parental helix
difference between type I and type II topoisomerases
- type I topoisomerases nick and reseal one of the DNA strands with no ATOP
- type II topoisomerases nick and reseal both DNA strands with ATP
when do replicator selection and origin activation occur
- replicator selection occurs in G1 = formation of pre-replicative complex
- origin activation occurs in S phase = unwinding of DNA and recruitment of DNA polymerase
what are replication origins controlled by
high levels of cyclin-dependent kinase (cdk) activity in S phase activates existing pre-RC but prevents formation of new pre-RCs
- G1 = low cdk = pre-c formation and no activation
- S1 = high cdk = no new pre-RC and activation
cdk levels in G1
- G1 = low cdk = pre-c formation and no activation
cdk levels in S1
- S1 = high cdk = no new pre-RC and activation
what enzymes are involved in finishing replication
- DNA polymerase and ligase close all but one gap
- RNA primer at end of chromosome is removed by RNase H ⇒ gap = 3’ overhang
what enzyme forms telomeres
telomerase
what sequence is added to extend the 3’
TTAGGG
structure of telomeres
ribonucleoprotein with intrinsic RNA component acting as template for telomere repeat sequences to be synthesised in step-wise process using reverse transcriptase = telomerase shuffle
what is the telomerase shuffle
- telomerase rna pairs up with existing telomere repeat + 3’ overhang
- telomerase uses this as template to add 3’ nucleotides on
- shuffles forward to synth next 3 nucleotides and again to bind to next 3 nucleotides etc
