chapter 13 Flashcards
chemical composition of DNA
DNA is a polymer of nucleotides
Purines and pyrimidines
antiparallel strands: direction is determined by sugar-phosphate bonds
phosphate groups connect the 3’ C of one sugar with the 5’ C of the next
One strand has a free5’ phosphate group — the 5’ end
The other chain has a free 3’ hydroxyl group— the 3’ end
Chargaff’s rule
In all DNA there is an equal amount of purines to pyrimidines
But the relative abundances of A & T versus G & C varies among species
X-ray diffraction data indicated that:
- the bases are on the inside of each strand
- The sugar-phosphate groups are on the outside of each strand
- the chains run in opposite directions—antiparallel
forces that keep them together
Hydrogen bonds between complementary base pairs hold the two strands of the DNA helix together
van der Waals forces occur between adjacent bases on the same strand
Three Steps in DNA Replication
Initiation: Double helix unwound, making two template strands using helicase (uses energy from ATP hydrolysis)
Elongation: addition of complementary base pairs linked by phosphodiester bonds
Termination: DNA synthesis ends when all DNA regions have been replicated
Start of DNA rep
DNA replication starts when a large protein complex (pre-replication complex) binds to a region called origin of replication (ori)
DNA polymerase requires a primer, a short starter strand—usually RNA
The primer is complementary to the DNA template and is synthesized by a primase
DNA polymerase then adds nucleotides to the 3’ end until that section is complete, and the primer is degraded
other proteins have roles in replication
single-stranded binding proteins keep the strands from getting back together
unzipping
At the replication fork, DNA opens up like a zipper in one direction
the leading strand grows at the 3’ ends as the fork opens
in the lagging strand, the exposed 3’ end gets farther from the fork, and an unreplicated gap forms
synthesis of the lagging strand occurs in small discontinuous stretches called Okazaki Fragments
each fragment requires its own primer
DNA polymerase III adds nucleotides to the 3’ end, until reaching the primer of the previous fragment
DNA polymerase I then replaces the primer with DNA
The final phosphodiester linkage between fragments is catalyzed by DNA ligase
Telomeres
Eukaryote chromosomes have repetitive sequences at the ends called telomeres
Eukaryote chromosomes
In humans, the sequence is TTAGGG-3’, repeated about 2,500 times
The repeats bind proteins that prevent the DNA repair system from recognizing chromosome ends as breaks
PCR Technique
The principles of DNA replication were used to develop the polymerase chain reaction (PCR) technique
An automated process make multiple copies of short DNA sequences for genetic manipulation and research (DNA amplification)
when the terminal Okazaki primer is removed, no DNA can be synthesized to replace it (no 3’ end)
The short piece of single stranded DNA is removed, and the chromosome becomes shorter with each replication
After many division, genes mat be lost and the cell dies (does this explain aging?)
Telomerase
Continuously diving cells, such as bone marrow stem cells, have telomerase, which catalyzes the addition of lost telomeres
Telomerase is expressed in most cancer cells and is important in their ability to keep diving. It is a target for anti-cancer drugs
Cells have 3 repair mechanisms
1.) Proofreading: DNA polymerase recognizes mismatched pairs and removes incorrectly paired bases. Catches 99% or more mismatches
2.) Mismatch repair: Newly replicated DNA is scanned for mistakes by other proteins and mismatches can be corrected
3.) Excision repair: Enzymes scan DNA for damaged bases—they are excised and DNA polymerase I adds the correct ones
PCR mixture contains:
- a sample of double-stranded DNA (the template)
- 2 artificially synthesized primers
- 4 dNTPs
- DNA polymerase that can tolerate high temps
- Salts and pH buffer
