microbial structure and function mi720 fall 2007 dna replication andrew j. pierce department of...

Microbial Structure and FunctionMI720

Fall 2007

DNA Replication

Andrew J. Pierce

Department of Microbiology, Immunology and Molecular Genetics

University of Kentucky

FEMS Microbiol Rev.2002 Nov;26(4):355-74.The bacterial replication initiator DnaA. DnaA and oriC, the bacterial mode to initiate DNA replication.Messer W.

E. coli origin structure

DnaA boxes are shown as red arrowsAT-rich regions are shown in yellow

Replication origins of different bacteria

FEMS Microbiol Rev.2002 Nov;26(4):355-74.The bacterial replication initiator DnaA. DnaA and oriC, the bacterial mode to initiate DNA replication.Messer W.

FEMS Microbiol Rev.2002 Nov;26(4):355-74.The bacterial replication initiator DnaA. DnaA and oriC, the bacterial mode to initiate DNA replication.Messer W.

5'-TTA/TTNCACA-3'

A-box: KD ~25 nM

initial unwinding: DnaA: 28 bpDnaA+SSB: 44-52 bp

Initial origin melting

Origin proteins

dnaA melt duplex

dnaB main replicative helicasecommitment step for replication fork formation

dnaC load dnaB

dnaG primase

FEMS Microbiol Rev.2002 Nov;26(4):355-74.The bacterial replication initiator DnaA. DnaA and oriC, the bacterial mode to initiate DNA replication.Messer W.

Initiating DNA replication

Annu Rev Biochem. 1995;64:171-200DNA polymerase III holoenzyme: structure and function of a chromosomal replicating machineKelman Z, O'Donnell M.

2 DNA polymerase molecules and 9 other accessory factors

E. coli DNA polymerase III holoenzyme

Principles of Biochemistry, 3rd EditionHorton HR, Moran LA, Ochs RS, Rawn DJ, Scrimgeour KGPrentice Hall 2002

replicationfork

replicationfork

Principles of Biochemistry, 3rd EditionHorton HR, Moran LA, Ochs RS, Rawn DJ, Scrimgeour KGPrentice Hall 2002

Replicating E. coli genome

Other E. coli DNA polymerases

DNA pol I remove RNA primers and join Okazaki fragments

DNA pol II aka: dinA (damage inducible), polBproofread terminal mismatchesextend terminal mismatches

DNA pol IV aka: dinB (damage inducible)extend terminal mismatches without proofreading

DNA pol V aka: umuD'2Ctranslesion synthesis

When Pol III goes wrong…mismatches

Mol Microbiol.2005 Oct;58(1):61-70DNA polymerase II as a fidelity factor in chromosomal DNA synthesis in Escherichia coliBanach-Orlowska M, Fijalkowska IJ, Schaaper RM, Jonczyk P

pol IV also used for template slippage

Trends Biochem Sci.2000 Apr;25(4):189-95Coping with replication 'train wrecks' in Escherichia coli using PolV, Pol II and RecA proteinsGoodman MF.

Bigger problems for DNA replication

abasicsite

UV 6-4 photoproduct UV cyclobutane dimer

SOS response

TTRecA

RecA *

RecA* LexA

Genetics, Vol. 158, 41-64, May 2001Comparative gene expression profiles following UV exposure in wild-type and SOS-deficient Escherichia coliCourcells J, Khodursky A, Peter B, Borwn PO, Hanawalt PC

uvrA nucleotideuvrB excisionuvrD repair

dinA = polB = pol IIdinB = pol IV

sulA block cell division

lexArecA

ruvA resistanceruvB to UV

umuC UVumuD'umuD mutagenesis

RecA*

1 2

3

Proc Natl Acad Sci USA2001 Jul 17;98(15):8350-4Roles of DNA polymerases V and II in SOS-induced error-prone and error-free repair in Escherichia coliPham P, Rangarajan S, Woodgate R, Goodman MF

Pol II helping out

Proc Natl Acad Sci USA2001 Jul 17;98(15):8350-4Roles of DNA polymerases V and II in SOS-induced error-prone and error-free repair in Escherichia coliPham P, Rangarajan S, Woodgate R, Goodman MF

Translesion synthesis

polV: umuD'2C

E. coli genome

Microbiol Mol Biol Rev. 2005 Sep;69(3):501-26Replication termination in Escherichia coli: structure and antihelicase activity of the Tus-Ter complexNeylon C, Kralicek AV, Hill TM, Dixon NE

Mol Microbiol. 1999 Mar;31(6):1611-8Termination of DNA replication of bacterial and plasmid chromosomesBussiere DE, Bastia D

Replication termination

TerF TerB TerC TerA TerD TerE

Tus - Ter

Mol Microbiol. 1999 Mar;31(6):1611-8Termination of DNA replication of bacterial and plasmid chromosomesBussiere DE, Bastia D

5'-TTAGTTACAACATNCA-3'

Tus - Ter and DNA polymerase III

Microbiol Mol Biol Rev. 2005 Sep;69(3):501-26Replication termination in Escherichia coli: structure and antihelicase activity of the Tus-Ter complexNeylon C, Kralicek AV, Hill TM, Dixon NE

Replication fork disassembly

Microbiol Mol Biol Rev. 2005 Sep;69(3):501-26Replication termination in Escherichia coli: structure and antihelicase activity of the Tus-Ter complexNeylon C, Kralicek AV, Hill TM, Dixon NE