Lecture 7b- Eukaryotic DNA Replication Flashcards
What is the number of clusters in the Eukaryotic replication?
20-50 replicons initiate simultaneously (euchromatin replicates first)
How many times does DNA replicate during the S phase?
Only once
In yeast cells what is the origin elements?
Autonomous replication sequence (ARS)
True/False: ARS is highly conserved consisting of 11 bp seq known as A domain
True
What happens when you have a mutation in the A domain?
Abolishment of ARS function - if mutation in other elements that are nearby the A domain, will cause reduction but not prevention of origin function
Define consensus sequence
A theoretical representative nucleotide (or amino acid) sequence in which each nucleotide (or amino acid) is the one which occurs most frequently at that site in the different sequences which occur in nature
What is the TATA box seq?
TATAAT (-10 region)
What is the Origin Recognition Complex (ORC)?
ORC is a protein complex that consists of 6 different proteins similar to the initial complex in E.coli
What does the binding of ORC indicate?
Binding indicates the site of an origin of replication
True/False: Most of the components within the ORC remains with the ARS
True
What does it take to initiate replication in eukaryotic cell?
When new proteins are added onto the ORC or change occurs in the ORC - DOES NOT depend on the formation of a new ORC-ARS association
What are some of the important proteins that bind onto ORC in the G1 phase to prepare for initiation?
Cdc 6 (Cell division cycle kinase) CDT1 Yeast MCM (mini chromosome maintenance proteins)
What is the role of Cdc 6?
Cdc6 is unstable having a half-life of 5 mins. Synthesized and bind to ORC during G1 phase and has ATPase activity needed for initiatrion. Binding of Cdc 6 allo0ws other proteins to bind
What binds to ORC after Cdc6 binds to ORC?
CDT1 and MCM (helicase)
What happens when Cdc6 and CDT1 are released from ORC?
MCM is phosphorylated
After the MCM is phosphorylated, what forms?
origin licensing (prereplication complex)
When the prereplication complex forms, what stage of interphase is the cell ready to go through?
S phase
ORC, CDC 6 Aand CDT1, MCM etc are all classified as what?
licensing factors
What happens to the licensing factors prior to replication and after replication?
Prior to replication, the licensing factors are in the nucleus, after replication these factors are inactivated or destroyed
Where can you find the newly synthesized licensing factors?
Outside the nucleus in the cytoplasm, they are not allowed to enter unless new replication round begins
When do the newly synthesised licensing factors enter?
Mitosis phase when the nuclear membrane breaks down
What are the three main polymerases in Eukaryotic cell DNA replication?
Poly alpha= DNA elongation + no proofreading ability
Poly delta= high level of proofreading ability, takes over the role of Poly alpha on the lagging strand 9elongation of Okazaki fragments and gap fills on the lagging strand)
Poly epsilon= Takes over Poly alpha on leading strand + proofreading
What two polymerases can take over the role of polymerase alpha?
Poly delta for lagging strand and poly Epsilon on leading strand
MCM=, CDC6/CDT1=, FEN1=Lig I =
MCM= helicase, CDC6/CDT1= Helicase loader, FEN1= endo/exonucleases a taht removes primer (sometimes need RNASE H1) , Lig I = ligase
What is equivalent to gamma clamp holder in POLY III of E.coli?
RF-C (Replication Factor C) is the clamp loader, comprised of 5 ATPase
What does RF-C recognize?
primer-template junctions and loads PCNA (proof reading cell nuclear antigen) there
What is the role of proofreading cell antigen (PCNA)?
The clamp _ equivalent to beta clamp (processivity)
What are Replication Protein A represent?
SSB proteins - maintains single-stranded parts
What performs the same role as the coupling template unwinding function as DNA Poly III?
C-M-G complex (c= CDC45, MCM, G= GINS), Coordinate the leading and lagging strand so that synthesis is simulatnous (leading strand is slowed down to match lagging)
How is genetic information lost over time in replicating ion of linear molecules?
When removing the final RNA fragment - there is no 3’OH available onto which dna Poly I can bind to and fill the gap - 3’ overhang in the leading strand
Where can you find telomere?
At the end of the 3’ overhang which is G rich
What recognizes the telomere (G rich sequence)?
Telomerase
How does telomerase add on to the 3’ overhang?
Using an existing RNA template within the enzyme, telomerase extends the telomere region by one repeat unit. Telomerase has reverse transcriptase ability.
What happens after telomerase adds an additional repeat to the parental strand?
A primer is added at the end for DNA polymerase alpha to elongate from the primer In this way the original information at the ends of linear chromosomes completely copied in the new DNA
Define the role of telomerase
Enzyme that repairs and replicates the ends of chromosomes - a ribonucleoprotein (protein-RNA complex)
What happens to the length of telomere in yeast and animal germ cell line that is different from that of human somatic cells
Yeast cells and animal germ cells have mechanisms to maintain telomere length within a limited range
Human somatic cells - telomere length increases early in embryonic development and shortens with aging
Where is telomerase activity generally found in?
Yeast, germ cell, and embryonic cells and cancer cells
True/False: Aging cells replicate in the absence of telomerase activity
True: As a consequence leads to shortening of chromosome ends and loss of genetic information
After many generations, descendent cells will inherit defective chromosomes
What happens when the cell inherit defective chromosomes?
Cells eventually withdraw permanently from cell cycle and stops diving - Replicative cell senescence
Telomeres are bound by what protein complex?
Shelterin -telosome Protein Complex
In addition to Shelterin, what other thing do mammalian telomeres contain?
Nucleosomes that show histone modifications characteristics of heterochromatin domains. Subtelomeric regions are high methylated - negatively regulate telomere length and telomere recombination
