L5: DNA Replication Flashcards
Eukaryotic Cell Cycle
- S phase or DNA Replication
Replication
- carefully regulated by the timing of the cell cycle and critical cell cycle “checkpoints”
Watson-Crick semiconservative hypothesis
- one of each parental DNA strands will be present in each of the two newly synthesized daughter dsDNA
conservative vs. semiconservative DNA replication
- conservative:
- both parental DNA strands are conserved in the daughter DNA
- semi-conservative:
- only one of the two parental strands is conserved in the daughter dsDNA
Conservative DNA replication
- both parental DNA strands are conserved in the daughter DNA
Semiconservative DNA
- only one of the two parental strands is conserved in the daughter dsDNA
DNA replication is ______
semi-conservative
5 Features of DNA replication
- DNA replication starts from a replication origin
- DNA replication is a DNA template-dependent polymerization process that needs RNA primers
- DNA synthesis is always 5’-3’, so replication of dsDNA has to process in opposite directions (bidirectional replication)
- DNA is synthesized continuously (leading strand), as well as discontinuously (lagging strand-synthesized as Okazaki fragments)
- DNA replication is very accurate and fast
Direction of DNA synthesis is ______
- 5’-3’
- the 3’ hydroxyl group of the existing DNA is where the incoming nucleotide will be added
DNA synthesis
- substrates: Deoxyribonucleoside triphosphates
- chemistry: 3’ OH attacks phosphate on incoming nucleoside triphosphate
Origin of replication
- prokaryote:
- there is one single origin per genome (or plasmid), also called a replicon
- eukaryote:
- have multiple origins in each chromosome (10,000 in human genome)
The parental dsDNA needs to be opened to become two ssDNA at the _____ _____
replication origin
Replication of a circular bacterial DNA occurs from _____ _____
one origin
In _____, there are many origins of replication per chromosome
eukaryotes
DNA replication is _____
bidirectional
DNA replication is bidirectional
- REPLICATION FORK
- the place where dsDNA “melts” into two ssDNA, there are 2 forks per origin
- replication process at both ends of the replication fork
- LEADING STRAND
- strand that is synthesized continuously
- LAGGING STRAND
- strand that is synthesized discontinuously, resulting in Okazaki fragments
Replication fork
- the place where dsDNA “melts” into two ssDNA, there are 2 forks per origin
Leading strand
- strand that is synthesized continuously
Lagging strand
- strand that is synthesized discontinuously, resulting in Okazaki fragments
Since DNA synthesis is always 5’, one daughter strand is synthesized _____ and the other is synthesized _____
continuously, discontinuously
Consequences of replication fork structure
- one DNA strand (leading strand) grows continuously
- one strand (lagging strand) is synthesized in short pieces (Okazaki fragments) that must be joined together
- DNA lagging strands must be initiated repeatedly by RNA priming (primase can initiate an RNA chain)
Key enzymes and accessory proteins involved in DNA replication
- helicase
- DNA topoisomerase
- primase
- DNA polymerase
- RNase H
- DNA Ligase
- ssDNA binding protein (SSB)
- sliding clam protein
_____ unwinds dsDNA to create ssDNA
Helicase
Unwinding of the double helix at replication fork causes _____ of the DNA helix
supercoiling
DNA topoisomerase
- removes supercoils by creating a transient break in one or two strands of DNA, which releases the tension and allow DNA to return to normal helix (10bp per turn)
_____ is an RNA polymerase
Primase
Primase
- an RNA polymerase
- synthesizes short RNA primers (5-10 nucleotides long) to help the initiation of DNA replication
SSB
single strand DNA binding protein
SSB
- bind and stabilize ssDNA
DNA polymerase
- both prokaryotic and eukaryotic cells have multiple DNA polymerases
- DNA polymerase III is the major E. coli DNA replication enzyme
- has 3 major enzyme activities
Major enzyme activity of DNA polymerase
- 5’ -> 3’ polymerase activity–for SNA synthesis
- 5’ -> 3’ exonuclease activity–for removal of RNA primers
- 3’ -> 5’ exonuclease activity–for repairing mistakes
RNase H
- specifically degrades RNA in the RNA:DNA hybrid
- removal of the RNA primer by RNase H and the 5’-3’ exonuclease activity of DNA polymerase
DNA ligase
- creates a phosphodiester bond between an adjacent 5’ phosphate and 3’ OH
- fills the gaps between Okazaki fragments to make a continuous DNA molecule
Sliding clamp protein (Beta clamp)
- helps polymerase to bind to the template strand rapidly after each round of lagging strand synthesis
Proof reading
- DNA polymerase has proofreading function (3’-5’ exonuclease activity), which allows it to detect mistakenly incorporated nucleotides, remove them, and replace with the correct nucleotides
- good but not perfect, reason for “spontaneous” mutations in the genome and why organisms have kept evolving
Summary: DNA is synthesized in a _____ manner
semi-conservative
Summary: DNA is synthesized _____
bidirectionally
Summary: At each replication fork, DNA is synthesized continuously on the _____ strand and discontinuously on the _____ strand
leading, lagging
Summary: _____ is used as primer
RNA
Summary: RNA primer is removed later and replaced by _____
DNA
Cancer
- accumulation of multiple mutations in two types of genes and the break-down of regulatory mechanisms of cell growth
- two types of genes involved in cancinogenesis are: oncogenes and tumor suppressor genes
- mutation in both the proto-oncogene and tumor suppressor gene are usually needed to cause cancer in which also why it is so difficult to treat cancer
- tumor suppressor genes are genes for which the loss of function causes cancer.
- can encode transcription factors cell-cycle regulators,phosphates, etc…
- tumor suppressor gene products are so often inhibitors of cell proliferation
Two types of genes involved in cancinogenesis are:
oncogenes and tumor suppressor genes
_____ _____ genes are genes for which the loss of function causes cancer.
tumor supressor genes
tumor suppressor gene products are so often inhibitors of ____ _____
cell proliferation
