Enzymes involved in DNA replication Enzyme Role Helicase or DnaB ...
Enzymes involved in DNA replication Enzyme Role Helicase or DnaB Unwinds DNA, breaks H-‐bonds to separate the two strands -‐ Requires energy – hydrolyses NTPs to NDPs + Pi -‐ Arrives in the initiation complex, present throughout replication Single stranded binding Stabilises strands and keeps strands apart protein (SSBP) -‐ Binds to single-‐stranded DNA Topoisomerases Untangles and relaxes the DNA Type I – cut one strand of backbone, allowing other strand to pass through gap, then reform -‐ Energy from potential energy stored in the twisted DNA Type II – cuts both strands and introduces negative supercoiling, then reform -‐ Energy source is required Primase (type of RNA Lays down short RNA primers for DNA pol III at initiation and the lagging strand polymerase) -‐ Provides the 3’OH required by DNA polymerase DNA polymerase III Copies the bulk of the DNA – adds nucleotides to synthesising strand DNA polymerase I Removes primer and fills in the gaps in the lagging strand Ligase Seals sugar-‐phosphate backbone -‐ dNTP – nucleoside triphosphate for DNA (deoxyribose) -‐ NTP – for RNA – nucleotide triphosphate (ribose) Polymerases -‐ All polymerases (DNA and RNA) have 5’ → 3’ polymerase activity o Make new DNA strand in 5’ → 3’ direction from a DNA template o Can ONLY add to existing 3’OH o dNTP substrate -‐ DNA polymerases need a primer o Short fragment of single-‐stranded nucleic acid bound to template o Provides a 3’OH to make the next addition § Cancer drug – omit the 3’OH and terminate elongation -‐ RNA polymerases o Make RNA copy from a DNA template in 5’ → 3’ direction o No primer required, use NTPs as substrate -‐ All DNA (not RNA) polymerases have a 3’ → 5’ exonuclease activity – proof reading o Cleaves backbone, removes recently added nucleotide if not complementary to template o DNA pol I (but not III) also has a 5’ → 3’ exonuclease activity to remove primer -‐ DNA pol III – main replication enzyme o Catalyses formation of phosphodiester bond between 5’P on incoming, and existing 3’OH § Results in new nucleotide being added to growing chain, and removal of two phosphate groups from that new NTP/dNTP o Processivity due to donut-‐like pair of β subunits which clap the enzyme to DNA § Remains tightly associated with template through many nucleotide additions -‐ DNA pol II, IV and V – repair enzymes
DNA Polymerase I -‐ Discovery of DNA pol I – incorporation of radioactive [32P]dTTP into DNA o Assay found that the product DNA reflected the base composition of the template DNA, not the relative conc. of the 4 nucleotides o Proof that template was being copied, not just making a random DNA polymer -‐ Why DNA pol I is not the main replication enzyme o Doesn’t work fast enough o Not processive enough (falls off after 20 bases) o No effect on rate of E. coli replication Replication process -‐ Full blown replication occurs only once, just before cell division -‐ Replication stages – initiation, elongation, termination -‐ Strands always built from 5’ → 3’ direction o Add nucleotides to 3’ end of the template strand -‐ Leading strand (parent strand 3’ → 5’ orientation) o Synthesized continuously in 5’ → 3’ direction 1. RNA primase used at the beginning (only once) 2. DNA polymerase III adds bases continuously -‐ Lagging strand (parent strand 5’ → 3’ orientation) o Synthesized discontinuously in shorter multiple pieces (Okazaki fragments) o Direction of synthesis is opposite to the direction of the growing replication fork o Template must wrap around – approach DNA pol III assembly from the opposite direction 1. Primase reads DNA template and attaches a short complementary RNA primer § Each fragment requires a new primer 2. DNA pol III extends primed segments – forms Okazaki fragments 3. DNA pol I performs “nick translation” when Okazaki fragment is complete § Removes primer (using 5’ → 3’ exonuclease activity) § Fills in gap (using 5’ → 3’ polymerase activity and proof reading activity) § However, still leaves a break in the backbone 4. DNA ligase joins gaps together § Seals sugar-‐phosphate backbone by reforming the phosphodiester bond § Requires energy source – NAD in E. coli; ATP in higher organisms § DNA pol I cannot join the nick: • Relies on hydrolysis of pyrophosphate from dNTPs to form the phosphodiester bond • Not adding another nucleotide here
DNA Polymerase I -‐ Discovery of DNA pol I – incorporation of radioactive [32P]dTTP into DNA o Assay found that the product DNA reflected the base composition of the template DNA, not the relative conc. of the 4 nucleotides o Proof that template was being copied, not just making a random DNA polymer -‐ Why DNA pol I is not the main replication enzyme o Doesn’t work fast enough o Not processive enough (falls off after 20 bases) o No effect on rate of E. coli replication Replication process -‐ Full blown replication occurs only once, just before cell division -‐ Replication stages – initiation, elongation, termination -‐ Strands always built from 5’ → 3’ direction o Add nucleotides to 3’ end of the template strand -‐ Leading strand (parent strand 3’ → 5’ orientation) o Synthesized continuously in 5’ → 3’ direction 1. RNA primase used at the beginning (only once) 2. DNA polymerase III adds bases continuously -‐ Lagging strand (parent strand 5’ → 3’ orientation) o Synthesized discontinuously in shorter multiple pieces (Okazaki fragments) o Direction of synthesis is opposite to the direction of the growing replication fork o Template must wrap around – approach DNA pol III assembly from the opposite direction 1. Primase reads DNA template and attaches a short complementary RNA primer § Each fragment requires a new primer 2. DNA pol III extends primed segments – forms Okazaki fragments 3. DNA pol I performs “nick translation” when Okazaki fragment is complete § Removes primer (using 5’ → 3’ exonuclease activity) § Fills in gap (using 5’ → 3’ polymerase activity and proof reading activity) § However, still leaves a break in the backbone 4. DNA ligase joins gaps together § Seals sugar-‐phosphate backbone by reforming the phosphodiester bond § Requires energy source – NAD in E. coli; ATP in higher organisms § DNA pol I cannot join the nick: • Relies on hydrolysis of pyrophosphate from dNTPs to form the phosphodiester bond • Not adding another nucleotide here
