L11: Chromosome Replication and Organization

oriC

origin of replication: where initiation proteins bind. Contains an AT-rich repeat region, with an IHF site, and high and low DnaA affinity sites

DnaB helicase

protein in replisome that separates two parental DNA strands

DNA gyrase

protein in replisome that removes supercoils created by helicase

DnaN sliding clamp

protein in replisome that tethers polymerases to template DNA

SSB (single strand binding protein)

proteins in replisome that bind tightly to ssDNA to protect and stabilize it

DNA polymerase III

protein in replisome that synthesizes new DNA strands in the 5’ → 3’ direction

DnaA

ATPase in replication initiation that binds to specific sites (of nine nucleotides) and catalyzes unwinding of AT-rich repeats (13 nucleotides long)

IHF

protein in replication initiation that binds and bends DNA to facilitate ATP-DnaA binding

DiaA

protein in replication initiation that helps DnaA bind to low-affinity sites

Step 1 of replication initiation

DnaA binds ATP-DnaA; ATP-DnaA, DiaA and IHF bind to oriC and create open complex where the AT-rich region has been unwound

Step 2 of replication initiation

DnaC loads Helicase DnaB onto the ssDNA region. This step is dependent on the interaction of this complex with DnaA. DiaA and DnaC leave oriC.

DnaC

protein involved in replication initiation that loads helicase DnaB onto the ssDNA region

Step 3 of replication initiation

DnaB helicase expands the region of ssDNA. Primase DnaG binds to helicase and synthesizes the first primers

DnaG primase

protein involved in replication initiation that binds to helicase and synthesizes primers

Step 4 of replication initiation

DnaN sliding clamp and polymerase III loaded onto the DNA, DNA synthesis begions

SeqA

protein that inhibits reinitiation of replication by blocking access to the newly replicated oriC. Binds to hemimethylated GATC sites within oriC and prevents ATP-DnaA from binding. Process generates time lag between initiations in e.coli

Dam methylase

protein that methylates entire chromosome on both strands on A residues within the sequence 5’-GATC-3’

how does SeqA replication inhibition work?

1. immediately after replication initiation, the origin GATC sites are hemimethylated (new strand not yet methylated)

2. SeqA binds to hemimethylated GATC sites within oriC and prevents ATP-DnaA from binding

3. SeqA and Dam compete for binding to origin sites

4. eventually Dam accesses GATC sites and converts them to the fully methylated state

5. SeqA can no longer bind; ATP-DnaA can rebind to initiate another round of replication

Hda

Protein that binds to clamp and stimulates ATP hydrolysis by DnaA, which makes DnaA incapable of initiating another round of chromosome replication. This is called RIDA: Replicative Inactivation of DnaA

RIDA: Replicative Inactivation of DnaA

Replication inhibition where Hda binds to clamp DnaN, and turns ATP-DnaA into ADP-DnaA. Inactivated DnaA unable to initiate replication. Gradually, ATP-DnaA is replenished by other processes. Creates a time lag between initiations

Nucleoid

condensed organized form of chromosomal DNA, featuring loops of DNA bound by SMC dimers. Chromosomes oriented such that origins are at poles, and termini are near midcell

SMCs (structural maintenance of chromosome proteins)

condense chromosomal DNA by capturing and stabilizing loops of DNA. These complexes help with chromosome compaction and organization. Genes usually essential for viability