Lecture 18 - DNA replication Flashcards
What type of replication is DNA replication?
DNA replication is semi conservative
In what direction does DNA replication occur?
DNA synthesis occurs in a 5’ to 3’ direction by formation of phosphodiester bonds
Where are phosphodiester binds added to?
hydroxyl ends
What can only be added to 3’ end?
Bases
What are anti-parallel?
DNA strands are anti-parallel to one another
What is highly conserved?
Key components of the E.coli and human replication forks are highly conserved
What are Okazaki fragments?
- Okazaki fragments are short DNA segments synthesized on the lagging strand during DNA replication
- They are necessary because DNA polymerase can only add nucleotides in the 5’ to 3’ direction, but the lagging strand is oriented in the opposite direction.
- After synthesis, the fragments are joined together by DNA ligase, forming a complete, continuous DNA strand.
What initiates DNA synthesis?
DNA synthesis is initiated by creating a Replication Fork, where the DNA strands are separated
What does DNA Helicase do?
separate the paired strands of DNA
Why can both new strands not be synthesized continuously?
The antiparallel orientation of parental strands and unidirection orientation of new DNA synthesis (5’ to 3’) means that both new strands cannot be synthesized continuously
- the template strands for Leading Strand and Lagging Strand synthesis are orientated antiparallel to one another at the Replication Fork
Describe complementary strand synthesis for the leading strand
Leading Strand Synthesis is continuous and occurs 5’ to 3’
Describe complementary strand synthesis for the lagging strand
Lagging Strand Synthesis is discontinuous and also occurs 5’ to 3’
What is responsible for synthesizing DNA chain?
DNA polymerases - they can’t start making a DNA chain from scratch, but requires a pre-existing chain or short stretch of nucleotides called a primer
What are short RNA primers synthesized from?
Using template and NTPs by DNA primase
What occurs when the RNA primer is in place?
DNA polymerase ‘extends it’
What does Lagging Strand synthesis require?
Lagging Strand synthesis requires:
- DNA Primase
- DNA Polymerase
- Ribonuclease H
- DNA Ligase
to convert Okazaki fragments to a continuous strand of DNA
What is the role of DNA Primase?
makes RNA primer
What is the role of DNA Polymerase?
extends RNA primer - requires PRIMER-TEMPLATE junction
What is the role of Ribonuclease H?
extends RNA primer
What is the role of DNA polymerase?
Extends across gap
What is the role of DNA Ligase?
Seals the nick
What DNA Helicase do?
DNA Helicase uses ATP to separate parental DNA strands at the Replication Fork and move the Replication Fork forwards
What occurs when DNA replication goes wrong?
Mutations in genes encoding DNA helicase cause human disease such as Werner Syndrome a progeria (premature ageing) - mutations are autosomal recessive, occuring in RECQ helicase gene WRN
What is Bloom Syndrome?
a rare cancer syndrome caused by loss-of-function mutations in RecQ-family DNA helicase which maintains genome integrity
What is processivity?
Processivity in DNA replication refers to the ability of DNA polymerase to remain attached to the DNA template strand and synthesize DNA for an extended period without dissociating.
What is the processivity of DNA Polymerases greatly enhanced by?
The processivity of DNA Polymerases is greatly enhanced by their association with a SLIDING CLAMP
- A processive polymerase will stay bound to DNA for longer
What occurs regarding processivity in DNA replication?
Once the first step of DNA synthesis has been accomplished, interaction of enzyme with a Primer/Template junction is maintained and addition of further nucleotides is very rapid.
The Sliding Clamp (ATP-dependent) is positioned close to the Primer/Template Junction by a Clamp Loader
How does the Sliding Clamp work?
Sliding Clamps encircle the DNA like a nut on a bolt and help move DNA polymerase forward
The Human Sliding Clamp, Proliferating Cell Nuclear Antigen (PCNA) has a near-identical 3D structure to this E.coli protein
What are Single-stranded DNA Binding Protein (SSBs)?
Single-stranded DNA Binding Protein (SSBs) expose single-stranded DNA in the replication fork, making it available for templating synthesis of the new DNA strand and easing replication fork progression
Single-stranded DNA Binding Protein (SSBs) keep Replication Fork open & enhance processivity of DNA Polymerase
What does DNA topoisomerases do?
They prevent DNA from becoming tangled during DNA replication and enhance processivity of DNA polymerase
Explain the relaxation of superhelical tension that occurs as the DNA is unwound at the replication fork.
- Helicase unwinding of parental DNA strands at the Replication Fork introduces super-helical tension into the DNA Helix
- Tension is relaxed by DNA Topoisomerases which nick and reseal the backbone of the parental helix
What do Type 1 topoisomerases do?
Nick & reseal one of the 2 DNA strands, no ATP required
What do Type 2 topoisomerases do?
Nick & reseal both DNA strands, ATP required
Where does DNA replication start?
a single point called “origin of replication
How many specific DNA sequences recruit replication initiator protein?
- E. coli (1 origin)
- Yeast (600-700)
- Humans (+ 100,000)
What are yeast origins called?
Autonomously Replicating Sequences (ARS)
What are human origins called?
poorly understood sequences near to LMNB2, MYC –> also defined by chromatin structure (e.g. nucleosome-free)
How is the initiation of DNA replication in eukaryotes biphasic?
- Replicator Selector occurs in G1 phase - formation of a pre-Replicative Complex
- Origin Activation occurs in S phase - unwinding of DNA and recruitment of DNA Polymerase
What does temporal separation of these 2 events ensure?
Ensures that each origin is used and each chromosome is only replicated EXACTLY once per cell cycle
What is the Pre-Replicative-complex called?
(pre-RC)
Describe the steps in the assembly of the pre-RC (pre-replicative complex)?
- Origin Recognition Complex (ORC) binds to replicator sequence - e.g. ARS sequence in yeast.
- Helicase-loading proteins Cdc6 and Cdt1 bind to ORC
- The Helicase Mcm2-7 binds to complete formation of pre-RC
- the pre-RC is in its inactive state
What do high levels of cyclic-dependent kinase (Cdk) lead to?
High levels of Cyclic-dependent kinase (Cdk) activity in S0phase activates existing pre-RC but prevents formation of new pre-RCs
Describe how differing levels of Cdk (cyclic-dependent kinase) affects DNA replication
Cdk activity low - pre-RC formation allowed, but no pre-RC activity
Cdk activity high - new pre-RC formation inhibited, but existing pre-RC activation
What does a close relationship between pre-RC function, Cdk levels and cell cycle ensure?
Ensures that chromosomes are replicated exactly once per cycle
In what stages are Cdk levles high/low?
G1 (assembly phase of pre-RC) - low cdk
S/G2/M - high cdk
What is a problem when finishing DNA replication?
problem at chromosome ends
What does Ribonuclease H do?
removes RNA primers, further shortening the newly synthesized DNA strands at 5’ ends of chromosomes
What is the risk of chromosomes shortening?
Chromosomes shortening risks loss of valuable coding information
What does addition of TTAGGG repeats by telomerase compensate for?
It compensates for the loss of telomere sequences caused by RNA primer removal and prevents chromosome shortening
How does telomerase function?
Extended 3’ end DNA is now long enough to enable DNA Primase to bind and initiate new RNA primer synthesis which can then be extended as an extra Okazaki fragment by DNA polymerase
What does Telomerase contain?
Telomerase contains an RNA component that specifies telomerase sequences
What is a telomerase?
Telomerase is a ribonucleoprotein with an intrinsic RNA component that acts as a template on which telomere repeat sequences are synthesized in a STEP-WISE PROCESS -THE TELOMERASE SHUFFLE
- (Telomerase RNA has U not Ts)
- Telomerase RNA allows addition of multiple TTAGGG repeats to the 3’-OH at each telomere
Summarise DNA replication
- replication fork progression requires the combined activities of DNA polymerase, DNA helicase, SSBs, DNA primase, DNA ligase, DNA topoisomerase and Ribonuclease H
- high processivity of DNA polymerases results from their association with Sliding Clamps such as Proliferating Cell Nuclear Antigen (PCNA)
- initiation of DNA replication is biphasic and involves temporal separation of Replicator selection and Origin Activation under control of cyclin-dependent kinases (CDKs). Temporal separation ensures that DNA replication occurs exactly once per cell cycle.
- Telomerase synthesizes the repeated sequences at telomeres, which allow linear eukaryotic chromosomes to be replicated without loss of chromosomal DNA sequence information
What do SSBs (single-stranded binding proteins) do?
Preventing re-annealing
