Lecture 14 Flashcards
7 traits of replication in euks and proks
- complementary base pairing
- semi-conservative
- unwinds the helix and moves as fork
- bidirectional
- requires RNA primer
- discontinuous on lagging strand
- requires enzymes called DNA polymerase
Origin
sequence of DNA where replication is initiated
Single well defined sequence that serves as the origin (only one for the entire chromosome)
OriC
Where are eukaryotic origins found?
more variable (many on each chromosome) Location and sequence has been determined by genetic and molecular approaches
Replicon
unit of genome in which DNA is replicated. Each contains an origin for initiation of replication.
Bidirectional
Replication fork moves in two directions
Unidirectional
Replication fork moves one way
Replicon timing
they do not all start at the same time (early vs late replication)
How can we map eukaryotic origins?
2 Dimensional gels
1st dimension
Separates on size with electric current
2nd dimension
exaggerates shape due to high agarose % and ethidium bromide
How do we extract the DNA from the gel?
Southern blot with hybridization
ARS
yeast Autonomously Replicating Sequences
ARS’ contain _____ sequences
CIS acting
ARS” confer ______ to plasmids
autonomous replication
An ARS is a well defined ________
eukaryotic origin
ARS interracts with the protein complex known as..
ORC, origin replication complex
What type of protein does ORC have?
Trans acting protein
How do they find ARS’s
mutagenesis
Linker Scanning Mutagenesis
Disrupts CIS acting sequence but maintains SPACING
How many bp elements are found in ARS I
4 related 11 bp elements important for origin function
Element A contains…
ARS consensus sequence of 11 bp and is absolutely essential for origin function
Domain of DNA that is easily unwound
B domain (likely A-T rich)
What is an ORC?
a complex of 6 proteins that bind to an ARS
What does ORC do?
facilitates local unwinding at the origin with helicase activity
Where does the ORC sit?
on the ARS throughout the cell cycle until activated
What activates the ORC?
CDC 6
Proposed model of the ORC and ARS
B region unwinds, cell cycle trigger
player 1 after orc activation and CDC6
- RPA cellular replication protein A
What does RPA do?
3 subunit ss DNA binding protein
Also binds pol alpha
Player 2 after orc activation and CDC6
DNA pol alpha/primase
What does DNA pol alpha/primase do?
synthesizes 1st RNA primer
Synthesizes DNA on lagging strand
Player 3 after Orc activation and CDC6
- RFC, replication factor C
What does replication factor C do?
Binds 3’ end of nascent DNA strand
Player 4 after orc activation and CDC 6
- PCNA, proliferating cell nuclear antigen
What does PCNA do?
subunit of DNA pol delta
