MCB 250 – Replication Initiation and Telomerase

VCasts 25 and 26

How many initiation sites does E. coli chromosome have?

one → oriC

Which protein is oriC recognized by?

a specific initiator protein, DnaA

Does DnaA require energy?

yes

What does the binding of DnaA-ATP do?

it begins a series of events that leads to the establishment of replication forks

How controlled is replication initation?

tightly controlled

How long is the oriC region?

~250 bps

What is the oriC region composed of?

multiple DnaA binding sites next to an A=T rich region

Which sequence does DnaA-ATP recognize?

the A=T rich rigion

What is DnaA-ATP and what does it do?

a nucleoprotein complex that causes unwinding of the DNA

How does DnaA-ATP open up the A=T rich region?

DNA binding around ATP increases negative supercoiling which opens the region up

What are the steps of replication initiation?

1. DnaA-ATP binds to its recognition sites in oriC

2. DnaA-ATP causes unwinding of the DNA

3. DnaC loads DnaB (helicase) onto the ssDNA

   • one helicase on each strand

4. helicase travels 5’ to 3’ and recruits primase → initiates DNA synthesis

Draw a diagram for replication initiation. Include origin(s), leading and lagging strands, directionality, and label the first lagging strand Okazaki fragment(s).

Where does replication initiation only need to occur?

only on the lagging strand → the first lagging strand can act as the leading strand for the other replisome

What does the replisome MUST be capable of?

re-initiating the leading strand

Is cell division and DNA replication coordinated?

yes

What must be coordinated with DNA replication?

cell division

Why does DNA replication and cell division have to be coordinated?

each daughter cell needs to get a fully replicated chromosome

How does DNA replication and cell division achieve coordination?

Is DNA in E. coli methylated?

yes

What is Dam?

DNA Adenine Methyltransferase

What does Dam do?

it recognizes the sequence 5’-GATC-3’ which is found 1/256 base pairs, and methylates the A’s in the major groove

What does methylation tell us?

• which strand is old DNA → is methylated

• which strand is newly synthesized → is not methylated yet

Which strand – old or new – is methylated in DNA?

old strand

What is hemi-methylated DNA?

methylated old strand and non-methylated new strand

What is fully methylated DNA?

when both strands, old and new, are methylated

What is the speed of methylation in comparison to replication?

methylation is significantly slower than replication

How is replication initiation controlled?

• control of DnaA-ATP levels

• control of access to oriC

What is the rate-limiting step in replication initiation?

the binding of DnaA-ATP to oriC

How does DnaA-ATP regulate replication initiation?

• the concentration of DnaA-ATP is tightly controlled

  • the amount of DnaA is proportional to cell mass ∴ small (new) cells don’t have enough DnaA to initiate replication

  • DnaA-ATP is converted to DnaA-ADP as the replication fork passes

  • DnaA-ADP doesn’t work as an initiator and takes a long time to convert back to DnaA-ATP ∴ slowing down replication initiation

How does the oriC regulate replication initiation?

• oriC is inactive until it’s fully methylated

  • DnaA-ATP binds to fully methylated DNA

  • however, protein SeqA binds hemimethylated oriC (which is present before fully methylation) which prevents DnaA from binding ∴ slowing down Dam methylation and oriC activation

What does Seq A do?

it binds to hemimethylated oriC → prevents DnaA and Dam from binding → slows down re-initiation

How is replication terminated?

*not completely understood

1. the replisomes collide and everything gets unloaded

2. Pol I fills in any gaps

3. ligase seals any nicks

What does Topo IV do in replication?

it is required for recatenation

What are Type II topoisomerases used for in replication?

• to relieve positive supercoils in front of the replication fork

  • gyrase does this in E. coli

• to resolve catenanes

  • Topo IV does this in E. coli

Why does the replisome move more slowly in eukaryotes than in E. coli?

probably due to chromatin structure

How many origins of replication do eukaryotic chromosomes have?

multiple; perhaps thousands

Why do humans need more origins of replication than E. coli?

humans have 1000x more DNA than E. coli so replication needs to be initiated simultaneously at multiple origins on each chromosome

Draw the basic steps for eukaryotic replication.

Which phase does DNA get replicated?

S phase

What happens if DNA replication is incomplete?

leads to chromosome breaks during cell division

What happens when over-replication occurs?

leads to extra copies of regions of chromosomes

Does helicase need to be loaded in eukaryotic replication?

no, they’re already loaded and waiting for initiation to start

What is the biggest difference between eukaryotes and bacteria?

eukaryotes have linear chromosomes; bacteria have circular chromosomes

What does eukaryotes having linear chromosomes lead to?

the end replication problem

What causes the end replication problem?

eukaryotes have linear chromosomes

Does the end replication problem occur in bacteria?

no, because bacteria have circular chromosomes, and this problem only occurs due to eukaryotes having linear chromosomes

What is the end replication problem?

the synthesis of the lagging strand results in incompletely replicated DNA due to RNA primer being removed from the last Okazaki fragment but there isn’t a 3’ end to synthesize DNA to replace the removed RNA ∴ if this problem isn’t resolved, as DNA replicates, it gets shorter and shorter

How is the end replication problem compensated for?

telomerase performs “reverse transcription” and makes a DNA strand by copying an RNA strand

What are telomeres?

the ends of eukaryotic chromosomes

What are telomeres made up of?

tandem repeats of TG rich sequences

What do telomeres do?

they protect the ends of linear chromosomes from degradation and other bad things

What is telomerase?

• a ribonucleoprotein

• a “reverse transcriptase”

What does telomerase do?

• creates telomeres

• it makes a DNA strand by copying an RNA strand → adds dNTPs to the 3’ end of a primer but its template is RNA, not DNA → compensates for the end replication problem

How is telomerase different from other DNA polymerases?

telomerase uses RNA as template, whereas other DNA polymerases use DNA as template

Do higher eukaryotic cells express telomerase?

yes, but not sufficient enough to repair telomeres

What is senescence?

aging of cells → being unable to repair telomers

Why do cells experience senescence?

they don’t express levels of telomerase sufficient enough to repair telomeres

B

B → leading strands allows synthesis from 5’ to 3’

C. The oldest RNA primer is at W; the newest primer is at Z.