Bio 305: DNA replication and repair

Replication requirements

single stranded DNA template, RNA primer bound to template, dNTPs

dNTPs

DNA monomer

DNA synthesis happens

5’ to 3’

How is replication have high fidelity

1 error per every hundred million added, uses AT/CG bonding, active site shape, and polymerase proofreading

DNA replication steps

1. DNA melting 2. priming with RNA primer 3. 1st primer extension 4. 2nd primer extension 5. unwinding of supercoils 6. Removal of RNA primers 7. Joining of Okazaki fragments

Helicase

melts DNA at origin into ssDNA

Primase

synthesizes RNA primer

DNA pol a

1st primer extension on both strands

DNA pol E

2nd primer extension on leading strand

DNA pol d

continues 2nd primer extension

Topoisomerase

unwinds supercoils

Ribonuclease H

removes RNA primers

DNA ligase

joins Okazaki fragments

RPA

protects ssDNA

PCNA + RFC

clamp + clamp loader (hold DNA pol to DNA)

Telomere

protective cap

Where does DNA synthesis naturally shorten

5’ end

What strand is bad for telomere shortening

lagging

Telomaerase

ribonucleoprotein that produces/re-extends telomeres

Telomerase steps

1. Binds to ss3’ end 2. Addition of TTG via reverse transcription 3. Translocation and elongation 4. Repeat to lengthen 3’ end 5. Priming, synthesis, and ligation of okazaki fragments

Ligation

joining of 2 nucleic acid fragments into single chain by ligase

Repair mechanisms

base, mismatch, and nucleotide

Repair mechanisms processes

recognize damage and differentiate it, damage removal, gap filling, strand ligation

Most common repair mechanism

base

Base repair

fixes T-G mismatches and damaged bases, only removes base of nucleotide, before replication

Mismatch repair

fixes replication errors, removes nucleotides around mismatch, after replication

Nucleotide repair

fixes modified bases, removes around modified bases, before replication

Double stranded breaks

destructive breaks depending on different timing of the break

Non-homologous end joining (NHEJ)

before replication, homologous chromosomes, G0/G1

Homologous recombination (HR)

after replication, sister chromatids, S/G2

Which break is error prone (loss of info)

NHEJ

Rad51

mediates strand invasion by ss3’ end to create heteroduplex and D-loop