DNA replication Flashcards
Define cellular homeostasis?
finely tuned balance between minimising the generation and persistence of DNA mutations that affect genomic stability and those that do not affect genetic stability
Genetic diversity in the germ line is essential to allow ?
Genetic fitness (Darwin evolution)
chromosomal polyploidy or aneuploidy will cause what
diseases such as cancer
DNA is used to?
to encode hereditary information
Where does replication occur?
replisomes
Function of replisomes?
assemble at DNA loci start sites or ‘replication origin sequences’
Replication starts from ?
single site of origin along both leading and lagging strands
Once replication is complete?…..
replisomes dis-assemble
The replisome is composed of?
The core replication proteins (in all cell types) include a helicase, primase, DNA polymerases, sliding clamp, clamp loader, and single-strand binding (SSB) protein
Replication is divided into which three stages?
Initiation, elongation, termination
INITIATION?
Initiator proteins recognise and bind to site of origin on the DNA
ELONGATION?
Movement of replisomes and DNA synthesis on leading and lagging strands
Prokaryotic genomes are typically ? (3)
haploid, smaller in size and usually having a single circular chromosome found in the nucleoid
Eukaryotic genomes are (3) ?
diploid; bigger is size and organized into multiple densely compacted linear chromosomes found in the nucleus
Time taken for E.coli unwind and duplicate DNA, error rate?
1000 bp/s, one mutation for each 10− 6 to 10− 7 NT incorporated
In e.coli replication initiation involves which two structures? (2)
- Replicator sequence on the DNA called oriC
- An oriC binding initiator protein called DnaA
Describe the process in which a complex of supercoiled DNA wrapped around a central core- Initiation. Dependence and proteins required? (PROS)
5 monomers of the DnaA protein (52kD each), which recognises and binds 9bp repeats in oriC (consensus sequence of 5’-TTATCCACA-3’).
This ATP dependent process is facilitated by histone like HU or IHC proteins to help DNA bending.
Describe the formation of the open complex in initiation? And what two structures are required
(PROS)
DnaA proteins melt 3 tandemly repeated 13bp AT rich segments (consensus sequence of 5’-GATCTNTTNTTTT-3’ to the left of the oriC. This 45bp segment is insensitive to P1 nuclease in the double stranded state but can be cut by the P1 nuclease in the single stranded state.
Requires: ATP and DnaA
Describe the formation of the prepriming complex?(PROS)
DnaA protein complex recruits the DnaB6 and DNAC6 hexametric complex into melted region to form the prepriming complex. This allows 5 additional DnaA monomers to bind to the bound DnaA dimers. DnaC6 is then released.
DnaC6
an ATPase that promotes loading
What is the next step which occurs to the the pripriming complex? - initiation of pros
DnaB6, a helicase, unwinds the DNA in the prepriming complex in both directions. SSB (single strand binding proteins) and gyrase proteins are necessary for DnaB6 activity
helicase/gyrase function in initiation of pros
further unwinds the helix to allow entry of the RNA polymerase, which activates the primase to begin RNA primer synthesis.
Bidirectional replication of PROS by what process?
by the recruitment of the replisome complex
Dif between the origins of replication in eus vs pros, why? (2)
- eukaryotes have multiple ori sites that can be activated (‘fired’) simultaneously
- Differences in the number and complexity of the proteins required in this process.
Why?
To enable replication of the larger and more complex genomes.
What structure is formed in initiation of eus
two bi-directional replication forks
eukaryotic replicative helicase is ?
an 11-subunit complex referred to as CMG (an acronym of the three components required for helicase activity “C” = Cdc45, “M” = six Mcm subunits that form the ring shaped heterohexameric Mcm2–7 complex, “G” stands for the heterotetramer GINS complex ).
CMG stands for?
“C” = Cdc45,
“M” = six Mcm subunits that form the ring shaped heterohexameric Mcm2–7 complex,
“G” stands for the heterotetramer GINS complex.
activation of the ori sites triggers in both pros and eus
Replication
Activation is split into?- eu initiation
Two distinct phases: G1 AND S
what proteins are required when licensed in eus initiation
a process that requires the six-subunit origin recognition complex (ORC), along with Cdc6, Cdt1, and the Mcm2–7 complex.
The licensing process results in?- eus initiation
The licensing process results in two Mcm2–7 hexamers that encircle dsDNA and are positioned head-to-head (ie, the two hexamers bind via their N-terminal regions)
In proceeding to S phase what is required- eus initiation
several origin activation factors work together, along with two cell cycle kinases (CDK and DDK), to form the CMG complex
Result of proceeding to S phase- eus initiation
Cdc45 and GINS become tightly associated with Mcm2–7, and each CMG complex encircles only one strand of DNA
The CMG helicases function in initiation- eus
unwind DNA, forming the ssDNA needed for priming and replisome assembly
Following kinase activity?- eus initiation
Following kinase activity, replication factors assemble and unwind the DNA helix and culminate in the assembly of the eukaryotic DNA polymerases
DNA polymerises required for assembly- initiation of eus
Dpb11, Sld2, Sld3, and Sld7
What is and isn’t associated with the mature replisome?- eus initiation
Dpb11, Sld2, Sld3, and Sld7 is required for assembly but are not thought to be associated with the mature replisome, whereas Cdc45, GINS, and Pol ε, are associated with it.
Function of DDK and CDK kinase activity?- initiation in eus
DDK and CDK kinase activity completes assembly and primes the complex for helicase activation that is accomplished by Mcm10 and RPA.