Advanced Molecular cell Biology - Lecture 17

DNA replication is _____-____________

semi-conservative

what direction does DNA synthesis occur by phosphodiester bonds?

5’ → 3’

the primer strand is _____-________ to the template strand

anti-parallel

where are new bases added during DNA replication?

on the 3 hydroxyl end

how do bases get attached to the lagging strand :

• A replication fork is created where the DNA strand is separated via DNA helicase (separates paired strands of DNA)

• leading strand = continuous synthesis

• lagging strand = discontinuous synthesis

• Lagging strand uses Okazaki fragments, which are short sequences of DNA that are synthesised from an RNA primer and extended by DNA polymerase

• the RNA primer (A) is made via DNA primase towards the replication fork

• The Okazaki fragment is joined to the 5’ end of primer A (closer to the replication fork) via DNA polymerase and continues being formed in the 5’ → 3’ direction towards the previous RNA primer (B)

• then primer B is recognised and removed by ribonuclease H

• this leaves a gap in the DNA which is synthesised over by DNA polymerase and sealed by DNA ligase

DNA polymerase can't start making a DNA chain from scratch, but requires a?

pre-existing chain or short stretch of nucleotides called a primer

how are short RNA primers created?

DNA primase creates small primer regions from RNA, creating a DNA/RNA hybrid. This is done from free nucleotide triphosphates (NTPs) that are in the nucleus; it then binds at a specific region or sequence and adds RNA bases onto the DNA

What does the lagging strand require to convert Okazaki fragments into a continuous strand of DNA?

DNA primase, DNA polymerase, Ribonuclease H and DNA ligase

DNA helicase uses ______ to separate parental DNA strands at the __________ _____ and move the replication fork forward, it does this by forming a loop around each DNA strands at the replication fork, pulling the strands the separate directions

ATP, replication fork

Mutations in genes encoding DNA helicases cause human diseases such as?

werner syndrome: a progeria (premature aging)

describe the nature of werner syndrome mutations:

autosomal recessive, occuring in RECQ helicase gene WRN

describe bloom syndrome:

A rare cancer syndrome caused by loss-of-function mutations in a RecQ-family DNA helicase, which maintains genome integrity

The processivity (how efficient, the number of consecutive reactions without letting go of its substrate) of DNA polymerase is greatly enhanced by its association with?

a sliding clamp

describe what the sliding clamp does in DNA synthesis:

It is ATP-dependent and is positioned close to the primer-template junction by a clamp loader, which signals to DNA polymerase to add multiple nucleotides onto the DNA at a time

what kind of structure does the sliding clamp form around the DNA?

encircles the DNA like a nut on a bolt and helps to move DNA polymerase forward

the human sliding clamp, __________ ______ _______ __________ (PCNA) has a near-identical 3D structure to E.coli protein

proliferating cell nuclear antigen

Single-stranded DNA Binding Proteins (SSBs) do what?

Exposing single-stranded DNA in the replication fork makes it available for templating synthesis of the new DNA strand and eases replication fork progression, ensuring the strands don’t attempt to bind to one another, forming hairpin structures. This enhances processivity

what do DNA topoisomerases do?

prevent DNA from becoming tangled during DNA replication and enhance processivity of DNA polymerase

Helicase unwinding of parental DNA strands at the Replication Fork introduced?

superhelical tension into the DNA Helix.

how is superhelical tension into the DNA helix by helicase unwinding at the replication fork relaxed?

by DNA Topoisomerases, which nick and reseal the backbone of the parental helix

what do Type 1 topoisomerases do?

Nick and reseal one of the 2 DNA strands; no ATP required, this helps with unwinding at the replication fork

what do type 2 topoisomerases do?

nick and reseal both DNA strands, ATP required, this helps with supercoiling

what is the single point where DNA replication starts called?

origin of replication

what happens at the ‘origin of replication’?

Specific DNA sequences recruit replication initiator proteins

How many origins of replication do E.coli have?

1 (origin onC)

how many origins of replication do yeast have?

600-700

how many origins of replication do humans have?

more than 100,000

What are the yeast origins of replication called?

autonomously replicating sequences

initiation of DNA replication in eukaryotes is?

biphasic

what are the 2 phases of initiation of DNA replication in eukaryotes?

• Replicator selection occurs in the G1 phase (formation of a pre-Replicative complex (pre-RC))

• Origin activation occurs in S phase (unwinding of DNA and recruitment of DNA polymerase)

Why is it important that the 2 phases of DNA replication in eukaryotes are temporally separated?

ensures that each origin is used and each chromosome is only replicated exactly once per cell cycle

describe the events of phase 1 of DNA replication:

• Origin Recognition Complex (ORC) binds to the replicator sequence (e.g ARS sequence in yeast, not known in humans)

• Helicase-loading proteins Cdc6 and Cdt1 (targets of CDK) bind to ORC

• The Helicase Mcm2-7 (exists as a dimer) activates and binds to either side of the replicator sequence next to ORC, to complete formation of the pre-RC complex

High levels of what in S-phase activate the existing pre-RC but prevent the formation of new pre-RCs?

cyclin-dependent kinases (Cdk) activity

describe the events of phase 2 of DNA replication:

• CDK increases in concentration

• levels peak in S-phase

• binds to Cdc6 and Cdt1 and activates them

• Replication machinery starts to unwind the DNA

Close relationships between pre-RC function, Cdk levels
and cell cycle ensure that?

Chromosomes are replicated exactly once per cell cycle

when finishing DNA replication what is the problem?

At the chromosome ends, on the lagging strand, when you have the RNA primer at the very end, there is no polymerase to synthesise over the gap, as there is nothing upstream, leading to single-strand overhangs at the end of the lagging strand and one on the leading strand where it started.
Ribonuclease H removes the primer, which shortens the newly synthesised DNA strands at the 5’ ends of chromosomes, which causes risk of loss of genetic info.

how does DNA replication overcome the issue with single-stranded overhangs when finishing replication?

By forming telomeres, telomerase does this, which allows DNA primase to bind and initiate new RNA primer synthesis, which can then be extended as an extra okazaki fragment by DNA polymerase

how does telomerase know to add the telomere?

recognises the stop codon

what is telomerase?

A ribonucleoprotein with an intrinsic RNA component that acts as a template. (This recognises the TTA stop codon and adds the correct complementary bases to the single-stranded overhang)

Telomere repeat sequences are synthesised in a step-wise process, called the Telomerase Shuffle. It moves along the strand and rebinds at the start of the newly synthesised TTA and then adds TTAGGG; in summary, telomerase RNA allows the addition of multiple TTAGGG repeats (hundreds or thousands) to the 3’-OH at each telomere