Ch 11 Lecture (DNA Replication) Flashcards
initiation
recognition of the origin of replication by the replicon
elongation
replication of the parental duplex by the replisome
replisome
assembles at an origin of replication
elongates
joining/termination
completion of replication process, includes separation or joining of daughter duplexes
DNA polymerases synthesize DNA in
semiconservative replication and DNA repair
All DNA polymerases synthesize
5’ to 3’
some repair polymerases function as
independent enzymes
replicases are incrporated with other enzymes into a large complex called the
replisome
E. coli polymerases
Polymerase III is the primary replicase
Polymerase II is required to restart the replication fork after it is stopped by damage
Polymerase I is involved in both error repair and replication
Polymerases IV and V are error-prone polymerases that allow bypass around DNA damage
replicases usually have nuclease activity, meaning they have a
3’ to 5’ proofreading function
DNA polmerase I
First DNA polymerase to be characterized
Two parts
Klenow fragment
Polymerase and 3’5’ proofreading function
Small fragment
5’3’ exonuclease function
Excises 10 bases at a time
Free base pairing between incoming dNTP and parental strand would allow for
mismatches
how do mismatches happen?
Twisting of the helix allows for nonstandard pairs such as C=T
Unique four nucleotide combinations called tautomers also allows nonstandard pairs such as C=A
High-fidelity DNA polymerases have a precisely constrained … that favors binding of standard base pairs
active site
Rate of incorporation of incorrect nucleotides is …X slower than correct nucleotides
10,000
DNA polymerases can also differentiate between
rNTPs and dNTPs
A sugar with a 2’-OH cannot be easily accommodated in
the nucleotide-binding pocket
processivity
The ability of an enzyme to perform multiple catalytic cycles with a single template instead of dissociating after each cycle
Frameshift fidelity is increased by enzyme processivity, especially in homopolymeric regions
many DNA polymerases have a structure that resembles a
right hand
palm
primary elements of the polymerase catalytic site
fingers
binds to incoming dNTPs and moves the correct dNTP into close contact with the polymerase catalytic site
thumb
Maintains the correct position of the 3’-OH and also a strong association between the polymerase and the parental strand to facilitate processivity
metal cations in the palm domain
One metal ion reduces the affinity of 3’-OH for its hydrogen
More nucleophilic
Other metal ion stabilizes the negative charges of the β- and gamma-phosphates of the incoming dNTP and the departing pyrophosphate
the palm domain is composed of
a β sheet and contains the primary elements of the catalytic site
Also contains two divalent metal cations that interact with the 3’-OH of the primer and the correct paired dNTP