Module 7 Flashcards
DNA replication relies on the ____ of DNA strands, which can come apart and serve as the template for daughter strands
complementarity
Products of DNA Replication
Two semiconserved daughter strands
Conservative DNA Replication Model
Both parental strands stay together after replication
Semiconservative DNA Replication Model
Double-stranded DNA contains 1 parental and 1 daughter strand following replication
Dispersive DNA Replication Modele
Parental/daughter DNA segments are dispersed
Meselson and Stahl Experimental Results
Used heavy N isotopes to initially grow bacterial colony, after 1 generation they observed that the DNA was “half heavy”, confirming the semiconservative theory
Replication in bacterial chromosomes begins at the single ____
origin of replication (oriC in E. coli)
oriC contains 3 types of functionally important sequences:
- Dna boxes (sites for Dna protein to bind, causing strand to bend)
- AT-rich regions (sites where strands separate)
- GATC methylation sites (DNA adenine methyltransferase, prevents premature replication)
At bacterial replication fork, ____ separates DNA strands while ____ alleviates supercoils forming upstream
DNA helicase, DNA gyrase (aka topoisomerase II)
Single-strand Binding Proteins
Bind to separated DNA strands to keep them apart, serve as placeholders for new strand
RNA Primers
“Prime” DNA synthesis, synthesized by primase enzyme. Leading strand has one continuous while lagging has fragments
DNA Polymerase III
Responsible for most DNA replication, composed of 10 different subunits (forming DNA Polymerase III holoenzyme)
Unusual enzymatic requirements of DNA polymerase include:
- Replicating only in the 5’-3’ direction (while moving in the 3’-5’ direction)
- Require RNA primers on nucleotides in order to synthesize a new strand
Leading Strand Synthesis
One RNA primer made at the origin, DNA polymerase III attaches nucleotides in 5’-3’ direction as it slides toward replication fork
Lagging Strand Synthesis
Synthesizes new strand in 5’-3’ direction, moves away from replication fork. Many RNA primers required, DNA polymerase III synthesizes small fragments using RNA primers (Okazaki fragments). Okazaki fragments have primers removes via DNA polymerase I, which fills the gap with DNA before ligase seals the strand
Primosome
DNA helicase and primase bind to each other to form complex, better coordinates actions of the two enzymes
Replisome
Primosome associates with 2 DNA polymerase holoenzymes (one for each strand) to form larger complex around replication fork
Nucleotides are connected via DNA polymerase catalyzeing the formation of an ____ bond between innermost phosphate of the incoming deoxyribonucleoside triphosphate and the 3’ -OH of the sugar of the previous deoxynucleotide
ester (leads to release of other to P’s)
Processivity
DNA polymerase remains attached to template strand as it synthesizes new daughter strand, due to the several holoenzyme subunits. B subunit forms dimer ring around strand “clamp protein” which freely slides along ds DNA
3 reasons for the fidelity of DNA:
- A-T, G-C stability
- Structure of DNA polymerase on active site
- Proofreading function of DNA polymerase (exonuclease activity)
Origin of Replication in Eukaryotes
Begins with assembly of prereplication complex (preRC), including the origin recognition complex (ORC), a 6 subunit complex acting as the first initiator to replication (MCM helicase completes DNA replication licensing to create fork, begin synthesis)
Primers are removed in eukaryotic cells using ____, which runs into primer of adjacent Okazaki fragment and “pushes” it into a short flap, which is removed by ____
DNA polymerase delta, flap endonuclease
DNA Polymerase alpha
Associates with RNA primase to form RNA/DNA hybrid primer
DNA Polymerase epsilon and delta
Exchanges primer on strands for DNA (epsilon on leading strand), (delta on lagging strand) in a process termed polymerase switching
