Lecture 12: Replication Initiation and Termination and Telomeres/Telomerase

How many replication forks are made at a single ori?

two replication forks

What is ori c recognized by?

a specific initiator protein (DnaA)

What must happen for DnaA ( initiator protein) to act as an initiator?

DnaA must be bound to ATP

What does the binding of DnaA-ATP begin?

A series of events that leads to establishment of replication forks

True or False: Replication initiation is tightly controlled.

true

What is the structure of oriC?

• Region is about 250 base pairs long

• 9-mer sites bind DnaA-ATP—causing the 13-mer (A-T rich DNA) region to melt

What is the process of DNA replication?

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

2. DnaA-ATP nucleoprotein complex causes unwinding of the DNA

3. DnaC loads DnaB (helicase) onto the ssDNA

4. Each helicase recruits a primase to initiate DNA synthesis

5. A clamp loader recognizes the 3’ end and loads a clamp on each strand

6. The two replisomes are now formed and proceed around the chromosome

7. Helicase travels on the lagging strand and recruits primase to lay down primers on the lagging strand

What does unwinding of the DNA cause?

local negative supercoiling

How many base pairs is the ori?

about 250 base pairs

What is the recognition site for a DnaA-ATP?

the ori

How does DnaA-ATP form an open complex?

by wrapping around the DNA such as histones would

True or False: There’s a helicase on the leading strand and one on the lagging strand.

true

What knocks DnaA-ATP off the strand?

helicase

What is really knocked off of DnaA-ATP?

a phosphate is knocked off the ATP—slowing down the process

When do tau proteins begin to interact with the helicase?

when a clamp loader recognizes the 3’ end and loads a clamp on each strand

True or False: The primase in each replisome can only prime the lagging strand.

true

True or False: The first lagging strand (Okazaki fragment) serves as the leading strand primer for the other replisome.

true

Why is the leading strand not synthesized as a single continuous molecule?

At some appreciable frequency, the replication fork will “collapse” and need to be reassembled well beyond the origin

True or False: DNA replication must be coordinated with cell division.

true

How is coordination with DNA replication and cell division achieved?

the DNA is methylated

How is DNA methylated?

DAM (DNA adenine methyltransferase) recognizes the sequence and adds a methyl group to the A’s (the methyl group protrudes into the major groove)

True or False: Methylation is slower than replication; it takes time for newly replicated DNA to become methylated.

true

What does methylation tell the cell?

• The DNA is newly synthesized

• Which strand is the newly synthesized one?

  • The one that’s not methylated

• Which strand is the old one?

  • The one that is methylated

• Hemi-methylated

  • Will eventually be converted to the fully methylated state

How is replication initiation controlled?

• control of DnaA-ATP levels

• control of the access to oriC

What is the rate limiting step in initiation?

the binding of DnaA-ATP to OriC

How is the concentration of DnaA-ATP tightly controlled?

• The amount of DnaA is proportional to cell mass — small (new) cells do not have enough to initiate replication

• As the replication fork passes, DnaA-ATP is converted to DnaA-ADP and it dissociates from its DNA binding sites

• DnaA-ADP doesn’t work as an initiator and is very slowly converted to DnaA-ATP

  • This is one of the ways a new round of initiation is blocked until the time is right

True or False: There are 11 GATC sites of methylation in Ori C.

true

What binds fully methylated DNA?

DnaA-ATP

What binds a hemi-methylated ori c?

the protein Seq A

True or False: Ori C is inactive until fully methylated.

true

True or False: Dam competes with SeqA for binding, but Dam eventually fully methylates the DNA; SeqA slows down methylation but cannot completely block it.

true

When does replication end?

when the two replisomes collide

What does type II topoisomerases do in replication?

decantenation

What is decatenation?

the process of separating interlinked DNA molecules

What does type II topoisomerases do?

Relieves positive supercoils in front of the replication fork

What can replication lead to?

catenated DNA molecules

What resolves the catenane?

the topoisomerase

True or False: Eukaryotic chromosomes have multiple origins.

true

True or False: The replisome of eukaryotes is functionally equivalent to that of E. coli, but the replisome moves more slowly in eukaryotes (30-50 nucleotides/sec).

true

Why does the replisome move slower in eukaryotes?

probably due to chromatin structure

True or False: Humans have a 1000 x more DNA than E. coli, so replication is initiated simultaneously at multiple origins on each chromosome.

true

How far are origins placed apart?

30 kb apart

What is important to know about DNA replication and regulation?

• During S phase, all of a eukaryote’s DNA must be replicated once and only once

• Incomplete DNA replication leads to chromosomal breaks at cell division

• Over-replication can lead to extra copies of regions of chromosomes

True or False: To replicate DNA, eukaryotes have the problem of disassembling all of the structures that compact the DNA into chromatin and, after replication has taken place, putting it all back together.

true

When are helicases loaded and activated?

helicases are loaded at the ori only in G1 and are activated only in the S phase

What is the end replication problem?

The last bit of DNA cannot be synthesized on the Okazaki fragment; it’s chemically impossible

What are telomeres?

the ends of the eukaryotic chromosomes

What do telomeres contain?

tandem repeats of TG rich sequences

What do telomeres do?

this structure protects the ends of linear chromosomes from degradation and other negative things

What is telomerase?

A reverse transcriptase that makes DNA by copying an RNA strand

What does telomerase use as a template?

an internal RNA template

True or False: Telomerase is a ribonucleoprotein.

true

True or False: As with all DNA polymerases, telomerase adds dNTPs to the 3’ end of a primer, but it’s template is RNA and not DNA.

true

Telomerase extends the 3’ end of the telomere so what can happen?

the additional 3’ end DNA can act as template for the new Okazaki fragment, and Okazaki fragment can be repaired

What type of overhang does telomerase form?

3’ overhang

What is senescence?

In higher eukaryotes, most cells don’t express levels of telomerase sufficiently to repair telomeres, so only a limited number of replication/cell division cycles can occur.

What human cells express telomerase and do not sensce?

germ and stem cells

What is the relationship between telomeres and cancer?

Cancer cells divide rapidly and most have acquired mutations that allow expression of telomerase

What is a potential target for anti-cancer drugs?

telomerase

True or False: Telomeres are special and critical for chromosome protection. Also, they contain silenced genes and are bound to the nuclear envelope.

true