Revisded October 17, 1998.
This is old Lecture 21
MCDB 2150 Lecture 22
Review of DNA Replication
Text Assignment: Chapter 11, Pages 298-317, 320-323 (The
section describing DNA recombination, pages 317-320, will be covered
in MCDB 3500. You do not have to learn it for this course).
Outline format: Because this lecture is supposed to be
primarily a review and because it moves rapidly through a large
amount of material, these notes are presented in outline form
only. Also, please note that except where labeled otherwise, most
of the descriptions are for bacterial systems. Eukaryotic DNA
replication is similar in principle, but differs in a number of
details, and is less well understood overall.
Templated replication.
- Sense and antisense strands
- Semiconservative replication
- Messelson-Stahl experiment.
- Sister chromatid labeling
Pattern of replication
- Replication forks
- Biirectional replication,
- Theta structure in bacteria
- Replicons
Initiation of replication
- Specific sequences at origin(s) of replication (9mers and 13 mers)
- Initiator protein (DnaA protein) binds at origin
- Helicase, ATP-driven opening of double helix (DnaB protein)
- Single stranded DNA-binding proteins (SSBPs).
- Topoisomerase I (gyrase) relieves twisting strain.
5' to 3' synthesis.
- Addition of new nucleotides occurs only at 3' ends of strands
- Hydrolysis of dNTP provides energy to add nucleotide to strand
- Leading strand, lagging strand
- Discontinuous replication of lagging strand
- Okazaki fragments
RNA priming,
- Deoxynucleotides can only be added to 3'-end of pre-existing strand
- New synthesis is primed with a short segment of RNA that is later removed
- Primase enzyme adds RNA primer
- Primasome complex (both leading and lagging strands).
DNA polymerase III
- Replisome complex
- Dimer plus many accessory protiens
- Loading function of gamma complex
- Clamping function of beta subunits
- dNTP's are added only at 3' ends.
- Concrrent synthesis on leading and lagging strands
Proofreading
- 3' to 5' exonuclease activity of DNA polymerases III and I.
- Polymerases can remove newly inserted nucleotides and try
again for a correct match.
DNA polymerase I.
- Discovered first, but not the primary enzyme for new DNA synthesis
- Probable has a major role in DNA repair.
- 5' to 3' exonuclease activity
- Important role in removal of RNA primer
- Primer ribonucleotides replaced with deoxyribonucleotides
Final steps
- DNA Ligase, joining of fragments.
- Topoisomerase II (cuts and reseals at termination of DNA synthesis).
DNA polymerase II
- Probably involved primarily in repair.
Eukaryotic DNA replication
- Polymerase alpha appears to be major enzyme.
- Polymerases beta and delta are also nuclear and probably involved
in repair.
- Polymerase gamma is mitochondrial.
- Multiple replicons in eukaryotic cells
- Histone synthesis tightly linked to DNA synthesis with immediate
formation of new nucleosomes.
Telomerase
- In a linear chromosome, DNA polymerase cannot replace primer
at 5'-end of lagging strand with DNA
- Telomerase uses its own RNA primer to generate special end
structures known as telomeres.
- Telomeres have characteristic repeated sequences (TTAGGG in humans)
- Telomeres and telomerase provide a mechanism for maintaining
full length ends of chromosomes.
DNA recombination (Pages 317-319) will be covered in MCDB
3500.