6 Principles of DNA replication
- Replication is semi-conservative
- Replication is initiated at specific sites
- Replication is typically bidirectional
- replication is semi-discontinuous
- RNA primases needed to start DNA replication
- nucleases, polymerases, ligases replace DNA primers with DNA and seal remaining nick
About sites of replication
- linear chromosomes can have multiple origins
- circular chromosomes have one origin
Replication fork
point where parental duplex separates and daughter duplexes form
Replication bubble
open DNA being replicated
Bidirectionality of replication
-replication occurs toward and away from replication fork but always in 5’-3’ direction
About replication semi-discontinuity
- leading strand synthesized continuously
- lagging strand discontinuously in opposite direction of replication fork
- Okazaki fragments produced
About primers
- leading and lagging strand need 10-13 BP RNA primers to start replication
- primers must be complementary to template and contain free 3’OH group
What is a nuclease
enzymes that hydrolyze the phosphodiester linkages of nucleic acids
Polymerases
-enzymes that catalyze template-dependent synthesis of DNA from its dNTP precursors
Ligases
enzymes that create phosphodiester bond between 3’ end of DNA segments and 5’ end of another
Domains of DNA Pol I.
- the DNA polymerase
- 3’-5’ proofreading exonuclease
- 5’-3’ exonuclease
Overall goal of exonuclease
enzyme that hydrolyzes only the phosphodiester bonds that are in terminal positions of the nucleic acid
Process of sealing nicks w DNA Pol I.
- At the pick , Pol I. degrades RNA primer in 5’-3’ direction, releases dNTPs
- simultaneously extends 3’ term with dNTP in same direction
- nick moves in 5’-3’ direction until all RNA removed
- Ligase seals fragments
DNA Pol Structure
- resembles right hand
- Fingers: where dNTP enter
- Thumb: holds template DNA in place
- Palm: DNA stand lays here, contains active site of enzyme
- Pol I. must be in closed form for rxn to take place
Quality control
- Pol. I relies on shape recognition
- G-C and A-T have shapes that fit well in closed form of active site
- incorrect pairs prevent full closure of Pol around DNA
Moieties
specific groups of atoms within a molecule responsible for characteristic chem. rxns if that mlc
About Pol. active site
- closed conformation allows important residues to contact sugar+phosphate moieties of incoming nucleotide
1. Asp residues (coordinate 2 divalent Mg++ atoms)
2. 1st Mg++ deprotonates 3’OH of growing strand, forms phosphodiester bond, releases pyrophosphate
3. 2nd Mg++ binds -ve charge phosphate groups in incoming dNTP, facilitates rapid release of phyrophosphate LG after addition of nucleotide
Steps of DNA Pol. Rxn
- Need primer strand, template strand, dNTPs
- dNTP added to 3’OH of primer strand causing growth - Pol. I moves forward to new 3’-term
- after dNMP added, new terminal BP occupies insertion site
- must be translocated to postinsertion site
- occurs by dissociation of enzyme from DNA followed by rebinding with terminal BP in postinsertion site
Pyrophosphate
phosphorous oxyanions with 2 P atoms in P-O-P linkage (diphosphate)
Insertion site
contains template strand nucleotide that will pair with incoming dNTP
Postinsertion site
- contains 3’-OH group of primer strand
- 3’OH attacks alpha and beta phosphates of incoming dNTP, results in addition of dNMP to primer strand + release of pyrophosphate
Translocation process of DNA Pol
- can slide b/c of several + charge Lys residues in palm domain
- interact with phosphodiester backbone
Processivity #
- the average # of nucleotides incorporated before enzyme dissociates from DNA
- low processivity used for a few bases (Okazaki frag.)
- high processivity used for long fragments
- Pol I has low #
- Pol III has high #
3’-5’ exonuclease activity (DNA Pol)
- when incorrect pair, Pol will fray DNA by 4 nucleotides
- places incorrect dNMP in 3’-5’ exonuclease active site, cleaves incorrect dNMP
- one Mg++ deprotonates H2O to form OH- nucleophile which mediates hydrolysis of incorrect dNMP (leaves free OH)
- another Mg++ promotes departure of incorrect dNMP LG from growing strand
