DNA Replication Making copies

overview DNA strands used as templates for replication are called PARENT strands These strands are ANTI-PARALLEL as they run in opposite directions (5’ to 3’ and 3’ to 5’) Newly formed DNA strands are called DAUGHTER strands DNA replication is semiconservative SEMICONSERVATIVE Replication: a mechanism of DNA replication in which each of the two strands of the parent DNA is incorporated into a new double-stranded DNA molecule Discovered in 1958 by Meselson and Stahl using “heavy” Nitrogen (15N)

Phase 1: Initiation Certain enzymes recognize specific areas of 100-200 base pairs called REPLICATION ORIGINS Since eukaryotic DNA is so long there are many origins on a DNA strand DNA Gyrase unwinds the DNA ahead of DNA HELICASE which “unwinds” the strand at the beginning of the fork by breaking the hydrogen bonds between complementary base pairs This creates a Y-shaped structure called a REPLICATION FORK

Tension, Attraction and proteins Oh my! As the fork separates from the origin in both directions (REPLICATION BUBBLE), tension builds on the single strands TOPOISOMERASES are enzymes that cut the strands to help unwind the strand and relieve tension Once this is accomplished the cut sections of the strand are reconnected by the same enzyme The complimentary bases on the parent strands want to reform their Hydrogen bonds thus SINGLE- STRAND BINDING PROTEINS (SSBP) bind to each parent strand to prevent this from happening (prevent annealing) These proteins are removed when the DNA is copied and reused when needed

Phase 2: Elongation New strands are ALWAYS built 5’ to 3’ as DNA polymerases can only add nucleotides to the 3’ end The complementary strand is built with nucleoside triphosphates This means that the phosphate group on the sugar molecule has 3 phosphates attached to carbon 5 2 phosphates are cleaved off when the nucleotides are joined together The cleaving of the two phosphates provides the energy needed to “fuel” DNA replication

Phase 2: The Leading Strand DNA polymerase can only attach nucleotides to a 3’ end so how does replication begin? RNA PRIMERS are added by RNA PRIMASE at the beginning of the replication fork at the 3’ end of the template RNA primase builds a complementary primer to this 3’ end and thus the new daughter strand is built in the 5’ to 3’ direction Once the primer is in place DNA POLYMERASE III adds complementary nucleotides to the 3’ end of the daughter strand This strand builds TOWARDS the replication fork and continues as the DNA unwinds thus ONLY ONE primer is required at each bubble. This strand is called the LEADING STRAND

Phase 2: The Lagging Strand The other parent strand has its 3’ end near the replication fork thus DNA polymerase must synthesize the new strand AWAY from the opening bubble (helicase) when enough fork has opened RNA primase adds a primer near the fork DNA polymerase III adds nucleotides until it reaches the end of the segment When more of the fork opens RNA primase adds another primer and DNA polymerase III adds nucleotides until it reaches the last segment This strand is built in small “pieces” called OKAZAKI FRAGMENTS since this strand is not built in one continuous process it is called the LAGGING STRAND

The lagging strand continued Each segment of the lagging strand must be joined to the previous DNA POLYMERASE I cuts out the RNA primers and fills them in with DNA The last nucleotide of each segment is attached to the next segment by DNA LIGASE an enzyme that catalyzes the phosphodiester linkage

Phase 3: Termination DNA reforms its helical shape without the use of enzymes At the end of OKAZAKI fragments there will be a small gap from the RNA primer These unpaired DNA bases don’t mesh with the old strand, and fall off, leaving the new strand slightly shorter than the original Humans lose approx. 100 base pairs per replication Luckily the end of a chromosome there are telomeres (usually many G’s, in humans it is a repeat of TTAGGG)

Proof Reading As DNA polymerase III moves along it proof reads the base pairs to ensure they are complementary If there is a mismatch DNA polymerase III will not move forward, instead it will cut out the mismatch and replace it with the correct base pair Leads to an error rate of 1 in 1 000 000 base pairs A second complex comes through after DNA is made that contains proteins mainly DNA polymerase I, DNA polymerase II and DNA ligase DNA POLYMERASE II moves slowly along the strand and looks for shape distortions Cleaves out incorrect strand and replaces it with correct sequence DNA polymerase II also used for repair of DNA damaged by environmental causes