Module 8 Flashcards
What is Chargaff’s rule?
the balance b/w A-T & G-C in ALL organisms
- DNA structure is REGULAR + STABLE
What does X-ray crystallization tell us?
info abt the structure of DNA
- DNA is a double helix
- distance / angle between rings
What are the uses of nucleotides?
energy storage + use
Purines
A + G (2 rings)
Pyrimidines
T + C + U (1 ring)
RNA forms . . .
hairpins + loops
- can also have a hairpin double helix
- CAN have H-bonding by not consistent
- can form complex 3D structures
Explain what it means that DNA is semi-conservative.
because when replication occurs, only one of the parent strains if retained; the 2nd strand is formed using new bases
What is the sliding clamp?
assists DNA polymerase on both leading + lagging strand so that it can travel for a long time
makes replication more PROCESSIVE
All polymerases are joined at the . . .
clamp loader
What are the functions of DNA pol I?
- replaces RNA primers with DNA nucleotides on both strands
- involved in repair of DNA damage
- has 2 types of exonuclease activity
What are the functions of DNA pol III?
the MAIN polymerase in replication
- adds nucleotides to template strand during replication
- 3’ –> 5’ exonuclease activity only
Structure of DNA pol I
palm, fingers, thumb, insertion & post-insertion sites
only 1 subunit
Structure of DNA pol III
over 10 subunits
Function of DNA helicase
unwinds DNA helix; breaks H bonds
spinning motor of 6 subunits; hydrolyzes ATP + spins
Function of SSB
bind to single strand, prevent them from rejoining
also protect the single strand from NUCLEASE attack
Function of gyrase
binds ahead of helicase and reduces tension (relieves tensional strain created by DNA unwinding)
Function of primase
adds RNA primer
Function of ligase
seals nicks in lagging strand
DnaA
recognizes Ori sequence; opens duplex at specific sites in origin
What is the origin of replication?
sequence at which replication begins
Describe the initiation of DNA replication at the origin of replication
- replication bubble forms - at either end of this bubble is a REPLICATION FORK
- eventually, the forks move around & join up so that the entire chromosome is replicated
- more origins of replication are found in eukaryotes
Explain DNA replication on the lagging strand
1) RNA primer added to fragment 2
2) DNA pol III adds nucleotides in 5’ –> 3’ direction until it reaches fragment 1
3) sliding clamp runs loose, & a new one is added
4) DNA pol I replaces the RNA w/ DNA, adding to the 3’ end of fragment 2
5) DNA ligase forms a bond b/w the newest DNA & the end of fragment 1
DNA synthesis works by adding nucleotides at the 3’ end of the. . .
NEW strand
What is a telomere?
a sequence at the end of chromosomes (that protect the ends) on the lagging strand. The sequence at the very end cannot be replicated due to the absence of a 3’ end for DNA polymerase
Telomerase is an enzyme that adds telomeres to the ends & this prevents the shortening of the chromosomes
How is DNA fidelity maintained?
1) proof-reading ability
2) complementary bases (binding of nucleotides)
What are the 2 main active sites of DNA polymerase?
1) polymerization (adding nucleotides)
2) exonuclease activity (removing them)
What is a vector?
something that will hold a piece of DNA
What are resistance sites on a plasmid vector?
sequence(s) that allow us to select ONLY the bacteria that holds this plasmid & ALL of the other bacteria will die
What are restriction sites?
Some sequences where we can insert the DNA that we want
What are restriction enzymes?
1) recognize short PALINDROME sequences
2) cut a specific sequence
3) some generate sticky ends, others blunt ends
4) compatible ends can base-pair (& then joined by DNA ligase) to form recombinant DNA
What are the important features of E coli plasmid vectors for use in cloning DNA?
- origin of replication
- resistance sites
- restriction sites
restriction enzymes are actually. . .
VIRAL defense proteins from bacteria
What sections of a vector are needed for the expression of a protein?
1) bacterial promoter (P) or operator (O) sequences
2) poly-liker / multi-cloning sites (an area w many restriction enzyme sites)
3) selectable genetic marker (e.g. antibiotic resistance)
4) ori
5) gene encoding repressor that binds O and regulates P
You can express a protein for. . .
a) functional assays
b) in a model organism
c) in human cells in cell culture
Why are DNA primers used in PCR?
- more stable
- much easier to use in lab
- bc RNA degrades very easily
- we can buy them on the internet
