Cell Nucleus Flashcards
How does replication differ between prokaryotes and eukaryotes?
1) Initiates at many sites
2) One cycle completed before next starts
3) DNA is assembled into higher order chromatin structure
What specifies origin sites in; viruses, yeast, and higher eukaryotes?
1) origin sequence- DNA motif
2) Autonomous replicating sequence(ARS element). Consensus sequence + flanking sequences- DNA motif
3) Probability based in GC rich euchromatic regions. forms G quads, attracts and inherits specific histones and proteins
Why were viruses a model organism for DNA replication?
Single origin sequence and initiator protein
How were origin sequences investigated in higher eukaryotes?
1) SNS-seq Length based filtering of leading strands, purify with gamma exonucleases that can’t digest RNA primer
2) Init-seq: Transiently intiating replication and filtering for dsDNA. Use antibodies to filter for modified nucleotides
What factors determine higher eukaryotic replication origins?
- Proximity to promoter
- G rich sequences
- CpG islands
- G quadruplexes
- Histone modifications
- Open chromatin
- The associated protein complexes
How does replication avoid clashing with transcription?
Go in the same direction
How do centromeres avoid binding the same chromatid?
One centromere per chromatid
Why does heterochromatin tend to edges?
Pushed out of way, also helps define chromatin borders. But sometimes euchromatin will be close to pores for specific functions
How do you avoid knots in chromatin fiber?
Knots require accessible ends. Also binding to nuclear matrix
Does replication and transcription occur at the same time?
can be in similar regions but not in the same place
How do nucleosomes with centromere specific histones differ?
centromere specific H3 allows binding to kinetochore and recruitment and attachment of proteins related to or part of kinetochore
How do telomeres protect the DNA ends?
simple repeat sequence forms terminal hairpin loop, T-loop. Also forms telomere specialized heterochromatin using G quads which attracts sheltrin protein complexes
Why can yeast artificial chromosomes be made?
Their ARS can replicate without additional machinery in addition to coding for the centromere attach sites and replicating proteins
What stage and how does the pre-RC assemble?
- G2 phase
- all origins and backups pre-activated
1) Origin replication complex
2) Replication initiator cdc6
3) Recruitment of 1st Minichromosome maintainance(MCM) with Cdt1
4) MCM surrounds DNA
5) Recruit second MCM
How does replication firing occur?
- S phase
- Happens to both MCM
- Kinases form the CMG(cdc45, MCM, GINS) complex and start
- E-CDK2 dephosphrylates cdc6
- A-CDK2 dephosphorylates cdt1
- DDK phosphorylates MCM
- Something phosphorylates ORC
Which strand does helicase displace?
5’ end
What do the different DNA polymerases do?
alpha- priming delta- small steps, okazaki synthesis epsilon- leads, leading strand gamma- different, mitochondrial beta- beta patch, repairs and gap filling
Which polymerases can proof read?
delta and eta
What are the proteins needed for DNA replication?
DNA helicase RPA- single strand binding protein PCNA- sliding clamp RFC- loads and unloads PCNA Fen-1 - removes short primers Dna2- cut primer flaps DNA ligase I- Links okazaki fragments Topoisomerases- Deal with supercoils from splitting strands
How does replication start?
1) RPAs attach to ssDNA
2) alpha adds primers then falls off
3) RF-C attaches and recruits PCNA
4) delta attached and starts