Quiz 2 Flashcards
DNA replication
Duplication of a DNA molecule; synthesis; takes place in S phase of cell cycle
Semiconservative
Half old and half new
Conservative
Complete copy
Dispersive
Partially identical strands all mixed up
Meselson-Stahl experiment
Demonstrated that DNA replication in e. coli is semiconservative
E. Coli DNA grown in N15 nitrogenous base added to a N14 medium and then replicated then mixed with gravity to see what ratio and results showed N14/N14 N15/14; each new DNA molecule consisted of one old and one newly synthesized strand
Allows DNA strands to serve as template for synthesis
Complementarity
Origin of replication
DNA replication begins
DNA is replicated in a:
Bidirectional fashion; it proceeds in both directions from origin
Replication fork
Structure where DNA replication takes place
Replicon
A specific region of DNA molecule that replicates from single origin of replication
Ex: e. coli has a circular chromosome with only one origin of replication, so is one replicon
Eukaryotic chromosomes are:
Linear (not circular), much larger than bacterial chromosomes, and have multiple origins of replication
DNA strand
A chain of nucleotides
DNA polymerase
An enzyme (protein) that makes DNA by adding nucleotides to a DNA strand
Chain elongation
When nucleotides are added to a DNA strand
Primer
Strand that the DNNA polymerase adds nucleotides to
Chain elongation occurs:
In the 5’ to 3’ end (i.e. the growing end is the 3’ end)
Four requirements for DNA POLYMERASE
- An available OH group on a nucleotide (free 3’ end)
- Mg2+ ions that support polymerase by adding nucleotides to chain
- Nucleotides (i.e. building blocks; a.k.a. dNTPs)
- A template strand
Significant of 3’-OH group:
Primer where incoming nucleotide is added
Two DNA polymerases used for regular DNA replication:
- DNA polymerase I; removes primes and fills gaps
- DNA polymerase III; main polymerase used for leading and lagging strands; HOLOENZYME
Holoenzyme
Enzyme with several subunits
Core enzyme
Subunit that catalyzed DNA synthesis (2 or 3 per holoenzyme)
Sliding clamp loader
Subunit that helps load and remove sliding clamp onto template
Sliding DNA clamp
Subunit that helps to maintain binding to template strand
Processivity
Effectiveness of DNA polymerase
Unwinding the DNA helix
The OriC is a segment with five 9mers and three 13mers (AT rich, so easier to denature); DnaA binds to 9mers and a conformational change occurs, forming replication bubble–helicases then bind and unwind the helix, allowing the replication fork to move (SSBs then bind to prevent it from reanneling
Reducing tension
DNA grade makes cuts in the DNA to untwist the strands and patch them back together
Generation of RNA primers
Short strands of 10ish nucleotides RNA primers are synthesized by primase, an RNA polymerase, which then provides the free 3’-OH
Continuous vs discontinuous DNA synthesis
Takes place, antiparallel, in a 5’ to 3’ direction, so you have two antiparallel strands so synthesis must occur in opposite directions but the DNA polymerase only moves in one, so a leading strand is continuous and a lagging strand is discontinuous, containing Okazaki fragments (each with its own RNA primer)
One strand is made forwards and one is made backward (lagging strand is looped)
Exonuclease
Enzyme that removes nucleotides from the end of a DNA or RNA chain; 5’-‘3 kind that removes from the 5’ and and 3’-5’ kind that removes from the 3’ end
RNA primer must be removed
DNA polymerase I removes the primer using its 5’-3’ exonuclease activity
Gaps between new strands must be sealed
DNA polymerase I fills in the gap once the primer has been removed, and the fragments are joined by DNA ligase
Proofreading the synthesis
DNA polymerase has a 3’-5’ exonuclease activity that allows proofreading but it occasionally makes mistakes
Additional correctional support
DNA repair complex, MMR (mismatch repair) fixes mutations that DNA polymerase generates
Eukaryotic chromosomes
Have much more DNA, multiple origins of replication, contain both DNA and protein, linear, and ends called telomeres
Multiple origins
Allow genome to be replicated in a few hours, called replication bubbles
Eukaryotic origins of replication
Act as initiating locations for replication and control when it happens
Origin recognition complex
Protein complex that, in early G1 stage, binds to origin of replication and marks it as site, then additional proteins associated with ORC to create PREREPLICATION COMPLEX (pre-RC)
When DNA replication is initiated
In S phase, other proteins are activated and target the pre-RC, which results in unwinding of helix to form replication forks
Three DNA polymerase involved with DNA replication in eukaryotes
DNA polymerase alpha (RNA primers and initial synthesis, contains primase) is then switched out in polymerase switching with either epsilon (synthesizes leading strand) or delta (synthesizes lagging strand)
Eukaryotic Okazaki fragments
Much shorter (100-150 bps) as opposed to e coli (couple thousand)
DNA replication through chromatin
Chromatin proteins, called nucleosomes, must be removed for replication to occur and then added to new strand
Telomeres
Ends of linear eukaryotic chromosomes that consist of ling stretches of short repeating sequences to protect end of chromosome (3’ end is G rich and 5’ end is C rich); very end is SHORT SINGLE STRAND
How telomeres protect ends of chromosomes
T-loops, shelterin complex (stabilizes t-loop)
End-replication problem
Leading strand is okay but lagging strand is an issue because once primer is removed there is no available 3’-OH for DNA polymerase and telomeres would become shorter and shorter
Solved by telomerase (a ribonucleoprotein) that includes a piece of RNA called the telomerase RNA component (TERC) which functions as a guide i.e. base pair with telomare and template (reverse transcription, using RNA template to make DNA)
Telomerase RNA component
TERC - piece of RNA that serves as guide and template
Telomerase reverse transcriptase
TERT - portion of telomerase that syntehsizes DNA using RNA replace
Telomere issues
Cells that don’t have telomerase activity become shorter and shorter over time, causing senescence (cell stops dividing)
Cancer cells maintain telomere length
