Lecture 7 MBB 331

What are SSBs

coat single-stranded DNA

What are ssb mutants known as

quick stop mutants

How do SSB proteins bind DNA

Bind cooperatively (promotes the binding of another SSB to the adjacent ssDNA)

What are the functions of SSB proteins

Keeps parental strands separate to act as templates and protects them from endonucleases

Approximately how much DNA does a human ssBP cover

total of 8 nucleotides

What enzyme performs most DNA synthesis in E. coli

DNA Polymerase III holoenzyme

What model describes DNA Pol III function

trombone model of replication fork function

How are the leading and lagging strands coordinated

Both strands are produced by a single DNA Pol III holoenzyme

trombone model: how is this coordination accomplished

by looping the DNA of the lagging strand, brings together two points of polymerization

What connects DNA helicase to DNA Pol III

tau (τ) subunits

What else do tau subunits bind?

bind DNA Polymerase III core proteins

How many Pol III cores replicate the lagging strand

Two DNA Pol III core enzymes

What happens when DnaB is connected to DNA Pol III

DNA unwinds FAST (700 bp/s)

What happens when DnaB is not connected to DNA Pol III

DNA unwinding is slow (35 bp/s)

What stimulates DNA strand separation

Binding of DNA helicase to DNA Pol III holoenzyme

What happens periodically on the lagging strand

DNA primase associates with DNA helicase and synthesizes a new RNA primer

What happens immediately after a new RNA primer is synthesized

The sliding clamp loader assembles a sliding DNA clamp at primer:template junction

What happens next?

second lagging-strand recognizes the loaded sliding DNA clamp and initiates the synthesis of a new Okazaki fragment.

What happens when the first lagging-strand polymerase reaches the end of the Okazaki fragment

released from the sliding clamp

Which enzymes process Okazaki fragments

DNA ligase and DNA Pol I come along to seal the nick and remove the RNA primer

What is another job of the sliding clamp

Help DNA Pol I and ligase join the Okazaki fragments

Why must β clamps be recycled

Okazaki fragments outnumber clamps 10 to 1, so clamps must be recycled

What is the single replicator required for E. coli chromosomal replication

oriC

What DNA elements make up oriC

composed of four 9-mer DnaA-binding sites

What protein binds the 9-mer repeats

Multiple DnaA

What happens when DnaA binds oriC

leads to strand separation

Which protein stimulates formation of the replication bubble

HU (histone-like) protein

How many GATC sites are found in oriC

11 GATC sites that are methylated by Dam methylase

How many proteins participate in initiation

At least 10 different proteins

What do these proteins accomplish

they open the DNA helix at the origin and establish a prepriming complex

What is the first control mechanism

Inhibit replication initiation through methylation of oriC by Dam methylase

What is the second control mechanism

Inactivation of DnaA via ATP hydrolysis . DnaA-ADP bound to oriC cannot form the open complex

What is the third control mechanism

Conversion of DnaA-ADP to DnaA-ATP is slow

What is the fourth control mechanism

Increase in superhelical tension by RNA polymerase activity.

What is the end replication problem

there is NO 3'-OH primer cause NO DNA replication at 3' ends

How does replication end in circular genomes

Replication ends with RNA primer removal and replacement with DNA

Solution for end replication

telomerase + telomeres

What is telomerase made of

telomerase reverse transcriptase together with a noncoding telomerase RNA

How does telomerase work

extends the 3' ssDNA with dNTPs, using its internal RNA molecule as template

What happens after telomerase extends the chromosome

extended 3' ssDNA is filled by RNA priming and DNA synthesis

Why is a 3' overhang left

Removal of the RNA primer leaves a short 3' overhang that is bound by telomere to protect the ends

Why is telomerase important in cancer

Activation of telomerase prevents the loss of telomeres

What is the initiator protein

DnaA

What is DnaB

helicase

What is DnaC

helicase-loading protein

Why is it called the prepriming complex

next step is to form the first primer

Step One

DnaA binds in order to kink it

Step Two

the kink causes AT bonds to break and separates the double helix

Step Three

DNAB needs to be loaded by DNAC creating the prepriming complex

Step Four

Primase is encoded which primes Okazaki fragments to build lagging strand

Primase is encoded by

dnaG

DnaG and DnaB together form

The primosome

What are the two jobs of the primosome

Primes Okazaki fragments and DnaB helicase moves on the lagging strand

replication initiation Steps

Helicase unwinds DNA.

Primase binds helicase.

RNA primer synthesized.

Polymerase binds.

Both strands are produced by

A single DNA Pol III holoenzyme

Coordination is accomplished by

Looping the DNA of the lagging strand

Tau interacts with

DNA helicase and DNA polymerase

Two DNA Pol III cores are dedicated to?

Replication of the lagging strand.

trombone model: replication steps

Helicase unwinds.

Leading strand synthesized continuously.

Lagging strand loops.

Primase makes primer.

Clamp loader loads clamp.

Polymerase synthesizes Okazaki fragment.

DNA Pol I removes primer.

Ligase seals nick.

Where do replication forks meet

Halfway around the chromosome from oriC

termination Steps

Replication forks approach ter.

Tus binds ter.

DnaB stops.

Replication ends.

Topoisomerase II decatenates chromosomes.

What is a temperature-sensitive mutant

TS mutant is able to replicate at one region of a temperature range but not at another

What is a quick-stop mutant

replication immediately stops after shift to 40°C