Bio2000 Dna Replication Ppt

DNA Polymerase III 5’ 3’ 5’ 3’

3’ 5’

• Primary replicative DNA polymerase in E. coli • Catalyzes DNA chain elongation by the formation of phosphodiester bonds • Incoming dNTP is positioned for chain incorporation by H-bonding with template nucleotide • Can only extend chains from 3’OH termini, cannot initiate synthesis of new chains • Catalyzes leading and lagging strand syntheses

DNA Pol III Catalyzes Phosphodiester Bond Formation in a DNA Chain 5’PPP 5’

3’

P P P P

OH3’

OH 3’

P P P P P P P P P

5’

Some Important Features of DNA PolIII • Dimeric: one monomer associated with leading strand, other with lagging strand • 130 kD monomer (also known as α subunit) • Functions as part of DNA PolII holoenzyme complex which contains 10 subunits • Subunits ↑ reaction rate and processivity: reaction rate ~1000nts/sec processitivity 5 x 105 • Processivity is due to ‘sliding clamp’ of β subunit:

5’

3’

Helicase (DnaB) • Hexameric (6 x 50 kD) • Catalyzes unwinding of DNA duplex thereby exposing single stranded DNA • Different helicases with different polarities on two strands in duplex • ATP hydrolysis provides energy for unwinding

Single Stranded Binding Protein (SSB) • Tetrameric (4 x 19 kD) • Binds to single-stranded DNA and prevents duplex reannealing SSB

ssDNA

• >1000-fold affinity for single-stranded DNA compared to double-stranded DNA • Lowers DNA melting temperature, i.e., promotes DNA denaturation • Binding is cooperative resulting in coating of the single-stranded DNA:

Primase (DnaG) • 60 kD • Intiates Okazaki fragment synthesis from a single-stranded DNA template: Primase

RNA primer

• After addition of 10-12 ribonucleotides, primase is displaced by DNA PolIII which synthesizes DNA from the 3’OH group on RNA primer • Complexed with helicase in lagging strand

Replication Termination of the Bacterial Chromosome BIDIRECTIONAL REPLICATION Origin 3’ 5’

5’ 3’

ori

ter

Replication Termination of the Bacterial Chromosome • Termination: meeting of two replication forks and the completion of daughter chromosomes • Region 180o from ori contains replication fork traps:

ori

Chromosome

Ter sites

Replication Termination of the Bacterial Chromosome • One set of Ter sites arrest DNA forks progressing in the clockwise direction, a second set arrests forks in the counterclockwise direction:

Chromosome

TerB

TerA

Replication Termination of the Bacterial Chromosome • Ter sites are binding sites for the Tus protein • Tus: 35.8 kD DNA binding at Ter Monomer Tus DNA

Replication fork arrested in polar manner

Ter

• Tus may inhibit replication fork progression by directly contacting DnaB helicase, inhibiting DNA unwinding

Figure 2. Components and their functions in DNA replication fork Abbr.

Full Name (Composition)

Functions

ORC

Origin Recognition Complex: Mini Chromosome Maintenance Proteins (MCM, 6 homo-subunits), DNA2 helicase, large T antigen, DNA topoisomerases Replication Protein A (heterotrimer) DNA polymerase α (primase ) DNA polymerase δ or ε 3’ exonuclease (Pol δ or ε subunit) Proliferating Cell Nuclear Antigen (homotrimer) Replication Factor C (5 different subunits) RNase H(35), DNA2, FEN-1 nucleases Ligase I

Initiation / unwinding

RPA Pol α Pol δ or ε PCNA RFC Lig I

ssDNA binding RNA primer synthesis DNA polymerization / elongation Proof reading Sliding clamp for polymerase Clamp loader Okazaki fragment maturation Ligation of Okazaki fragments

DNA2 and FEN-1 nuclease complexes sequentially remove the “α-segment” of Okazaki fragment.

When FEN-1 flap endonuclease activity is inhibited, WRN and FEN-1 exonuclease resolve the secondary structure of the Okazaki fragment.

Ncl X NclX induces the collapse of the fork which leads to recombinational repair as an alternative for rescuing replication.

Figure 1. A cartoon that illustrates our central hypothesis.