DNA Replication and Recombination Lecture 4 DNA Replication and Recombination

DNA Maintenance Mutation rate are extremely low 1 mutation out of 109 nucleotides per generation

DNA replication Separation, Base pair

The Chemistry of DNA replication

DNA Synthesis by DNA polymerase

Nucleotide polymerizing enzyme, first discovered in 1957 DNA Polymerase Nucleotide polymerizing enzyme, first discovered in 1957

DNA replication with two forks

This Doesn’t Work!

DNA replication Fork

DNA Proofreading

Structures of DNA polymerase during polymerizing and editing E: exonucleolytic; P: polymerization

Why 5’->3’? The need for accuracy

Site-directed mismatch repair in eucaryotes In procaryotes, old DNAs are usually methylated on A while newly synthesized ones are not. So Cells can distinguish old and newly synthesized DNAs and mutate mismatches on new ones.

DNA Proofreading RNA usually doesn’t have this. Why? Pairing, correct nucleotide has higher affinity binding to the moving polymerase Un-Paired nucleotide is easier to be off before covalent ligation, even after binding. Exonucleotic proofreading Strand directed mismatch repair

DNA Primer synthesis On Lagging strand DNA primase

DNA Replication at the Lagging strand RNA primer is 10 nucleotides long

DNA ligase joins the 3 end of the new DNA fragment to the 5 end of the previous fragment This enzymes seals a broken phosphodiester bond DNA ligase uses ATP to activate the 5 end at the nick

Different Helicase on leading and lagging strand DNA Helicase DNA double helix are tightly coupled. High temperature is needed to break them (95oC) Different Helicase on leading and lagging strand

DNA Binding Protein (RPA) SSB: Single Strand DNA-binding Proteins, also called helix destabilizing proteins

Covers total of 8 nucleotides, DNA bases remain exposed DNA complex SSB Proteins DNA Covers total of 8 nucleotides, DNA bases remain exposed DNA complex

PCNA

Cycle of DNA Polymerase/Clamping Protein loading and unloading At the lagging strand

Protein machinery for DNA replication

A Moving Replication

Structure of the Moving Complex

DNA Topoisomerase prevent DNA tangling Reversible nucleases binds covalently to DNA backbone phosphate Breaking pdiester bond Reversible and reforms

DNA topoisomerase I

DNA topoisomerase II

Mammalian replication Fork (eucaryote, DNA polymerase (primase) a synthesize RNA/DNA, DNA polymerase delta is the real polymerase)

Summary DNA replication 5’->3’ DNA proof reading Lagging strand, back-stitching, Okazaki fragment Proteins involved: DNA polymerase, primase DNA helicase and single-strand DNA-binding protein (SSB) DNA ligase, and enzyme to degrade RNA DNA topoisomerases

DNA Replication in Chromosome

Initiating Proteins for DNA replication 1. Initiator protein, 2. helicase binding to initiator protein, 3. helicase loading on DNA, 4. helicase opens the DNA and binds to primase, 5. RNA primer synthesis, 6. DNA polymerase binding and DNA synthesis

The four standard phases of a eucaryotic cell DNA replication occurring at S Phase (DNA synthesis phase) G1 and G2, gap between S and M

Different regions of a chromosome are replicated at different times Arrows point to the replicating regions at different times

Some facts about Replication in eucaryotes Multiple replication origins occurring inclusters (20-80) (replication units) Replication units activated at different times Within replication units, replication origins are separated 30,000-300,000 pairs apart. Replication forks form in pairs and create a replication bubbles moving in opposite directions Different regions on the same chromosome are replicated at distinct times in S phase Condensed Chromatin replicates late, while less condensed regions replicate earlier

Addition of new histones Chromatin assembly factors (CAFs) help to add and assemble new nucleosomes Newly H3 and H4 are acetylated on their N-terminal tails after incorporated into chromatin AG are removed enzymatically

Reverse transcriptase with RNA template to bind to DNA strands Telomerase Structure Reverse transcriptase with RNA template to bind to DNA strands

Telomerase and its function

Summary Specific DNA sequence determine replication origin, recruiting proteins to form replication machinery. relatively complex in eucaryotes Bacteria has single replication origin. Eucaryotes have multiple origins and less defined. Replication forks are activated at different times in eucaryotes Telomere and telomerase