DNA Replication Machinery I Flashcards
Where does replication take place in the cell cycle
S phase
Duration of replication
8 hours (DNA synthesised ~50bp/sec)
Replication machinery is comprised of
DNA Polymerase and additional proteins
How have cellular proteins responsible for initiation, elongation, and cell cycle control been identified
- mutants defective in replication
- molecular, biochemical, structural, cytological
Systems used to identify cellular proteins
- PRO E.Coli
- Viruses SV40 and Adenovirus
- EU Yeast, Xenopus, Human, Drosophilia
Semi-conservative DNA Replication
each molecule of DNA ends up with one conserved strand and one newly synthesised strand
DNA Polymerase 5’-3’ chain elongation
- DNA Polymerase synthesises DNA
- activity requires presence of all deoxyribonucleic triphosphates, Mg2+ ions, and 3’OH group paired to DNA template
- adds unpolymerised complementary deoxyribonucleic acids to the 3’OH end of the new DNA chain
- can only proceed in 5’ to 3’ direction
- incoming deoxyribonucleoside triphosphate loses the terminal two phosphate groups
Initiation site
- ## origins of DNA replication
Daughter cell synthesis
- using both of the exposed parental single strands as templates
- antiparallel structure of two strands duplex DNA
Describe the synthesis of the lagging strand
- discontinuously
- 5’-3’ direction
- series of short fragments; Okazaki
- back-stitching process
DNA synthesis is synthesised
semi-discontinuously
What enzyme joins the Okazaki fragments
DNA ligase
EU DNA and Chromatin
- EU and PRO DNA differ in that eukaryotic DNA is synthesized as Chromatin
- DNA is complexed with tightly bound proteins called histones
- DNA is wound around a disk-like shape formed by an octamer of histones -> creates nucleosome
- chromatin remodelling enzymes can alter number and distribution of nucleosomes
Nucleosome inhibition of replication machinery
- inhibit access of the machinery to the origin of DNA replication
- act as barriers to slow down the movement of DNA replication forks
Disruption of the chromatin structure
- by movement of the replication fork as DNA strands separate
- nucleosomes are reformed immediately following replication of DNA
Role of chromatin remodelling proteins
stimulate DNA replication
Describe the movement of replication forks
outwards from the origins of replication in opposite directions
Describe origin of replication in prokaryotes
- circular
- have one replication origin
- replication forks proceed outwards from origin in opposite directions until the entire genome is duplicated
Replication origins in large eukaryotic genomes
- multiple DNA replication origins
- replication is thought to proceed until it encounters a replication fork moving the opposite direction or until it reaches the end of chromosomes
Clusters of replication origins in eukaryotic genome
within clusters only one origin is activated when there are sufficient nutrients, but all origins are activated when the nutrient supply is deficient, in order to replicate the DNA as efficiently as possible
When are dormant origins activated
only when a problem arises that leads to the stalling of two converging replication forks, such that the interlying DNA is in danger of not being replicated
Essential elements of DNA replication origins
- binding sites for initiator/origin recognition proteins
- start site of replication
- elements for unwinding/distortion of DNA
Non-essential regulatory elements of DNA replication origins
- binding sites for activators/chromatin remodelers
- contribute to efficiency of replication
PRO E.Coli DNA replication origin
- well-defined at the nucleotide level
- can support replication of any piece of DNA containing it
- initiator protein DnaA binds and contains two sets of AT-rich repeats
