DNA Replication, Repair, 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 Replication at the Lagging strand

DNA Ligase

DNA Helicase DNA double helix are tightly coupled. High temperature is needed to break them (95oC)

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

SSB Proteins DNA

DNA Clamping Protein

Cycle of DNA Polymerase/Clamping Protein loading and unloading At the lagging strand (how about leading strand?)

Protein machinery for DNA replication

A Moving Replication

Structure of the Moving Complex

DNA winding

DNA topoisomerase I

DNA topoisomerase II

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

DNA replication in Bacterial Genome

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

Regulation for DNA replication In Bacteria, hemimethylated origins are resistant to initiation, delayed methylation leads to delayed initiation at the second phase Dam methylase

DNA replication in eucaryotes Multiple replication origin 50 nucleotides/second, autoradiography

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

The search and identification of DNA replication sequence ARS: autonomously replicating sequence Only histidine expression can help cells to survive

The origins of DNA replication on chromosome III of the yeast S The origins of DNA replication on chromosome III of the yeast S. cerevisiae

A close look at an origin of replication in yeast ORC: origin recognition complex B1, B2, B3: other regions binding to required proteins

The replication origins of human genes are more complex Even far distant DNA sequences could be important

Histone remains associated with DNA In vitro experiments DNAs with different sizes are replicated. Only the daughter DNAs replicated from parental DNA with histones showed histone binding

Addition of new histones Chromatin assembly factors (CAFs) help to add and assemble new nucleosomes

Bacteria DNAs are circular, not a problem There is a problem for eucaryote DNAs: ??? Hint: Telomere

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

T-loops at the end of mammalian chromosomes

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

DNA Repair Spontaneous DNA damage Pathways to remove DNA damage Damage detection The repair of Double-strand break DNA repair enzymes

Spontaneous Alterations of nucleotides Red: oxidative damage; blue: hydrolytic attack; green: uncontrolled methylation

Depurination and deamination

Thymine dimer

Mutation Generation passed on to daughter DNAs

Mutation Generation passed on to daughter DNAs

DNA Repair I

DNA Repair II

Recognition of unusual nucleotide By base flipping recognized by DNA glycosylase family

Emergency DNA Repair for Double helix break

DNA Repair Summary Spontaneous DNA damage: spontaneous alteration of bases, depurination and deamination, thymine dimer Pathways to remove DNA damage: base excision repair, nucleotide excision repair Damage detection: base flipping The repair of Double-strand break: nonhomolous end joining, homologous end joining DNA repair enzymes: heat shock proteins

DNA Recombination General recombination Site specific recombination

General DNA Recombination

Heteroduplex joint

General Recombination Two homologous DNA molecules cross over The site of exchange can occur anywhere A strand of one DNA molecule has become base-paired to a strand of the second DNA to create heteroduplex joint No nucleotide sequences are altered

The procedure of general recombination DNA synapsis: base pairs form between complementary strands from the two DNA molecules

The initial step for DNA recombination DNA Hybridization The initial step for DNA recombination

RecA protein-mediated DNA synapsis Rec A has multiple DNA binding sites, hence can hold a single strand and a double helix together Rec A is also a DNA-dependent ATPase

DNA Branch Migration

Holiday Junction for DNA recombination Exchange of the first single strand between two different DNA double helices is slow and difficult, then intermediate state Holiday Junction, then complete exchange

Holiday Junction for DNA recombination and its resolution

Summary for General Recombination General recombination allows large fraction of genetic information to move from one chromosome to another. General recombination requires the breakage of double helices, beginning with a single strand breakage. General recombination is facilitated by Rec A in bacteria and its homologs in eucaryotes. Holiday junction is the intermediate state of general recombination

Site-specific recombination Moves specialized nucleotide sequence (mobile genetic elements) between non-homologous sites within a genome. Transpositional site-specific recombination Conservative site-specific recombinatinon

Transpositional site-specific recombination Modest target site selectivity and insert mobile genetic elements into many sites Transposase enzyme cuts out mobile genetic elements and insert them into specific sites.

Three of the many types of mobile genetic elements found in bacteria Transposase gene: encoding enzymes for DNA breakage and joining Red segments: DNA sequences as recognition sites for enzymes Yellow segments: antibiotic genes

Cut and Paste Transposition DNA-only

The structure of the central intermediate formed by transposase (integrase)

Replicative Transposition

Retrovirus-based Transposition Retroviral-like retrotransposition

Reverse Transcriptase RNA

Non-retroviral retrotransposition

Conservative Site Specific Recombination Integration vs. inversion Notice the arrows of directions

Bacteriophase Lambda

Genetic Engineering to control Gene expression

Summary DNA site-specific recombination transpositional; conservative Transposons: mobile genetic elements Transpositional: DNA only transposons, retroviral-like retrotransposons, nonretroviral retrotransposons