Various types of machinery are needed to adjust the structure of chromatin rapidly!

*!

! A molecular motor! ! Needed for both replication and gene expression.! ! Hence, chromatin structure is dynamic.! ! Chromatin-remodeling complexes can decondense chromatin!

Covalent modications of histones regulate chromatin structure (histone code)!

! These modications function, in part, by recruiting other chromatin-remodeling complexes.!

*!

Covalent modications of histones regulate chromatin structure (histone code)!

*!

! These modications function in part by recruiting other chromatin-remodeling complexes.!

Can also modify core histones to help alter chromatin (e.g., HATs)! Still learning what the codes mean!

Interphase chromosomes contain both condensed and more extended chromatin!

! Euchromatinmore extended, higher gene expression! ! Heterochromatincondensed, low gene expression! ! ~10% of interphase chromatin! ! Located around centromere and telomeres! ! There are heterochromatin-specic proteins! Heterochromatin can spread to coding regions and silence the gene.!
!

*!

Barrier DNA prevents heterochromatin spread!

*!

Figure 5-29 Essential Cell Biology ( Garland Science 2010)

Maternal Gene Silencing!

*!

One must be silenced, so that expression in females and males is similar.! ! Selection appears to be random! ! Silenced X is passed along the cell lineage! ! Females are Mosaics!

Chromatin structure can be inherited (epigenetic inheritance)!

*!

! Specic histone modications are passed on.! ! DNA methylation patterns are passed on.! ! This process occurs during differentiation of cells (committed)!

Essential Cell Biology

Third Edition

Chapter 6 DNA Replication, Repair, and Recombination !

Copyright Garland Science 2010

Template-directed DNA Synthesis !

*!

Base-paring enables DNA replication !

Watson-Crick base pairing; A-T and G-C! new strand ! old strand !

Semiconservative Replication!

One intact parent strand is passed onto each daughter cell.! ! Thus, errors of DNA replication are apparent as mismatched base pairing!

Figure 6-4 Essential Cell Biology ( Garland Science 2010)

How to distinguish between these 3 possibilities ?!

Meselson and Stahl Experiment !

*!

15N!

14N!

~ 1.2 % heavier!

Meselson and Stahl Experiment !

*!

14N!

15N!

Meselson and Stahl Experiment !

15N!

14N!

(~ 1 generation)!

How to distinguish between these 3 possibilities ?!

14N! 15N!

Meselson and Stahl Experiment ! showed DNA replication is NOT conservative !

*!

15N!

14N!

Meselson and Stahl Experiment ! showed DNA replication is NOT conservative !

*!

14N!

15N!

14N!

How to distinguish between these 2 possibilities ?!

How to distinguish between these 2 possibilities ?!

*!

number of generations!

2! 3!

2! 3!

Meselson and Stahl Experiment ! showed DNA replication IS semiconservative !

*!

1st!

2nd!

3rd!

Meselson & Stahl (1958)!

DNA replication happens during S Phase!

*!

DNA replication begins at replication origins !

Typically have very specic sequences! A-T rich!

*!

A-T rich!

Need to break H-bonds !

Origin of Replication : Bacteria vs Human !

! Bacteria (~6 million bps) have single origin of replication in their circular genome.! ! Human (~3 billion bps) have ~10,000 origins.! ! Why so many origins?!

*!

bacteria !

human !

DNA synthesis starts at replication forks !

! Y-shaped structures (replication forks)! ! Bidirectional!

S-Phase ! Chromatin!

DNA polymerase is a core component of the replication machinery !

! Synthesizes new DNA using one of parental strands as template.! ! ! Catalyzes the addition of nucleotides to the 3-end of a growing DNA strand by forming phosphodiester bonds between the 3 hydroxyl group and the 5-phosphate group of the incoming nucleotide.! ! ! The nucleotides come initially as high energy nucleoside triphosphates (also a nucleotide).! ! provides the energy for polymerization.! ! Thus, DNA is synthesized in the 5-to-3 direction.! ! DNA polymerase remains bound to template strand and slides along the DNA.!

*!

Must have 3 hydroxyl to elongate the strand !! !

Figure 6-10 Essential Cell Biology ( Garland Science 2010)

ATP hydrolyzed to AMP can be used if more energy is needed !

*!

!G0 = 32.2 kJ/mol!

!G0 = 33.5 kJ/mol!

Figure 3-40 Essential Cell Biology ( Garland Science 2010)

inorganic ! pyrophosphatase!

Proofreading DNA Polymerase!

*!

Figure 6-14 Essential Cell Biology ( Garland Science 2010)

DNA polymerase is self-correcting !

! Makes only ~1 error every 107 bps.! ! This is beyond the predicted accuracy.! ! DNA polymerase has 3-to-5 proofreading activity.! ! Monitors new base-pairing.! ! Can correct mistake using a nuclease that cleaves the phosphodiester backbone.! ! Occurs during DNA synthesis.! ! Polymerization and proofreading are carried out by 2 different domains of DNA polymerase.!

Proofreading corrections are possible! only if DNA is synthesized 5-to-3 !

The DNA replication fork is asymmetrical !

Solution: DNA polymerase uses backstitching to piece together short strands of DNA called Okazaki fragments. !

! Answer: DNA polymerase uses backstitching to piece together short strands of DNA called Okazaki fragments. Thus, this lagging strand is made discontinuously. These short fragments are subsequently joined together.!

*!

Short RNAs are primers for DNA synthesis !

Q: Since DNA pol (polymerase) can only join a nucleotide to a base-paired nucleotide in DNA, how does it start?! ! A: It needs a primer nucleotide sequence.! ! This priming is done by the enzyme primase. This enzyme does not need a base-paired end.! ! Primase makes short stretches of RNA (~10 bps) ! primers for DNA synthesis.! ! !Primase is an example of an RNA polymerase-an enzyme !that makes RNA using a DNA template. This RNA/DNA !duplex is known as a heterodimer.!

*!

On the lagging strand, DNA is made in fragments !

*!

! On the leading strand, only one initial primer is needed.! ! On the lagging strand, many primers are needed.! ! ! The RNA primers are removed by a nuclease that recognizes the RNA/DNA heterodimer.! ! A DNA repair polymerase with proofreading then lls in the gap (end of Okazaki is primer).! ! The completed fragments are nally joined/sealed by DNA ligase. !

DNA replication requires the coordination of many proteins to form the replication machine !

*!

*!

For Lagging Strand!

{!

1.! Need to unzip DNA-helicase (uses ATP)! 2.! DNA polymerase! 3.! Sliding clamp-keeps DNA pol on DNA. Putting this on requires another protein-the clamp loader (uses ATP).! 4.! Need to stabilize ssDNA so it doesnt rehybridize and keep it elongated-singlestrand binding protein (SSBPs)! 5.! Primase, a nuclease (not shown here), DNA repair pol, DNA ligase!

DNA replication requires the coordination of many proteins to form the replication machine !

DNA replication requires the coordination of many proteins to form the replication machine !

*!

Telomerase replicates the ends of eukaryotic chromosomes !

*!

! Problem: DNA polymerase cannot synthesize DNA in the 3to-5 direction. And, at the ends of chromosomes there is no place to lay down an RNA primer. How are telomeres replicated?! ! Solution: Eukaryotes have special repetitive DNA sequences in their telomeres that recruit telomerase.! ! Telomerase is part protein and part RNA. It recognizes the repeats and adds more repeats every time a cell replicates its DNA.! ! ! Telomeres also identify ends of chromosomes rather than dsDNA breaks!

Telomerase linked to both cancer and aging.!

Lagging strand cannot be completed !

*!

Must have a base-paired residue with a 3 hydroxyl to be synthesized by the DNA polymerase! ! Primase requires ~ 20 base pairs to generate a 10 base pair primer! ! ! At some point, there is not enough room left on the template strand for the primase!

*!

"#
Figure 6-18 Essential Cell Biology ( Garland Science 2010)

Failure to repair DNA mistakes can have serious consequences !

! Can lead to permanent changes in the DNAmutations. !

Ex. Sickle Cell Anemia !

A DNA mismatch repair system removes replication errors that escape DNA polymerase proofreading!
! DNA mismatch repairthe backup system! !
! Fixes DNA mismatches left behind by replication machine.! ! Pretty effective (>99%), but not perfect! !

Because germline mutations result in an entire organism having mutation, protecting the germ cells from mutations is critical!
! ! ! ! ! Germ cellsthe reproduction cells! = sperm and egg ! (ex. genetic diseases like SCA)! Somatic cellsevery other cell in ! your body (ex. cancer*)!
Due largely to the accumulation of mutations over time. Anything that speeds up this process could be disastrous (ex. Mutation or deletion of DNA repair enzyme).! ! colon cancer in women!

*!

Mismatches must be repaired properly to avoid mutations !

*!

bad !

worse !

good !

! In eukaryotes, still not known how DNA repair machinery tells the difference between the 2 strands. New DNA might be nicked (ss breaks).!

*!
BAD!

Figure 6-21a Essential Cell Biology ( Garland Science 2010)

*!
BAD!

WORSE!

Figure 6-21b Essential Cell Biology ( Garland Science 2010)

*!
GOOD!

GOOD!

Figure 6-21c Essential Cell Biology ( Garland Science 2010)

DNA mismatch repair !

*!

Distorts dsDNA; hence can be recognized as different!

Spontaneous events that compromise DNA integrity!

*!

Figure 6-23 Essential Cell Biology ( Garland Science 2010)

Spontaneous events that compromise DNA integrity!

*!

Spontaneous events that compromise DNA integrity!

*!

If not xed, can lead to mutations !

Thymine Dimers can form as consequence of UV radiation!

*!

Figure 6-24 Essential Cell Biology ( Garland Science 2010)

Damaged DNA can repair itself using its backup copy !

! Can use complementary strand as template.! ! ! Since most DNA damage creates strange looking structures, easy to differentiate the two strands.! ! Proteins (nucleases) involved in Step 1 vary with different types of DNA damage.! ! Base Excision Repair (BER) System!

*!

What happens when both strands of DNA are damaged? !

! Can happen from ionizing radiation, replication fork errors, various chemicals and metabolites, etc.! ! Nonhomologous end-joining (NHEJ) is the most common mechanism to repair dsDNA breaks in somatic cells.! ! Usually OK since most of genome non-coding.! ! Quick and dirty.!

*!

Homologous Recombination !
! Can produce error-free repairs.! ! ! Involves using entirely separate DNA duplex (ex sister chromatids) to x dsDNA break.! ! Also used extensively to produce genetic diversity during meiosis (swapping between maternal and paternal chromosomes). ! ! Requires extensive stretches of sequence similarity (homology). But doesnt have to be absolutely perfect homology.! ! Donor DNA needs to be in close proximity following dsDNA break.! ! Versatile DNA repair mechanism. Highly conserved.!

*!

Homologous Recombination !

*!

Homologous recombination during meiosis !

! During meiosis, chromosomal crossovers lead to the exchange of genetic information.! ! During meiosis recombination preferentially occurs between maternal and paternal chromosomes rather than between newly replicated, identical DNA strands like when HR repairs dsDNA breaks.!
(meiosis-specic proteins) !

Homologous recombination during meiosis !

! Can lead to 2 different types of recombination.!

*!
! If heteroduplex has any mismatches, DNA can undergo mismatch repair.!

more common !

less common !

*!

*!

Gene Conversion !

Crossover !

Discovery of Genetic Transposition!

Jumping Genes! Transposons !

*!

Barbara McClintock! (1902 1992)!

Mobile Genetic Elements (Transposons) !

! jumping genes (molecular parasites ?)! ! Short specialized sequences of DNA that can move throughout a cells genome.! ! Can carry other genes.! ! ! Responsible for much more rapid evolutionary genetic changes.! ! Typically affect only that cell and its descendants.! ! ! Can be major cause of antibiotic resistant bacteria.!

*!

Mobile Genetic Elements (Transposons) !

*!

inverted repeats!

5---GACTGCGCAGTC---3!

Mobile Genetic Elements encode the components they need for movement !

*!

! Unlike HR, dont require sequence homology.! ! Contains ! !(1) Gene for transposase (catalyzes the movement of that element via specialized recombination)! !(2) DNA sequences that are recognized by its transposase.! ! ! Nearly half of human genome! ! is occupied by millions of ! !copies of various mobile ! !genetic elements!!!

Human genome contains 2 major families of transposable elements !

1.! DNA-only transposons!
!

*!

2.! Retrotransposons!
! ! ! Uses RNA intermediate! Unique to eukaryotes! Most common type!

! L1 element (LINE-1); 15% human genome! ! Alu sequence; ~1 million copies in our genome; dont encode their own reverse transcriptase! ! Both proliferated in primates relatively recently.!

Alu Sequence Distribution!

*!

Arthrobacter luteus restriction endonuclease! ! ~ 300 bps! ! Formed from the 7SL RNA component ! of the Signal Recognition Particle!

Viruses: the ultimate mobile DNA !

Figure 6-37 Essential Cell Biology ( Garland Science 2010)

Viruses: the ultimate mobile DNA !

! Essentially strings of genes wrapped in a protein coat. Very small.! ! Parasitesthey need to use cells machinery to replicate.! ! Often lethal (ex lytic) to cell.!

Retroviruses are found only in eukaryotic cells.!

Viruses commandeer the host cells machinery !

*!

This lysis can cause an immune response. !

Retroviruses make DNA from an RNA template using reverse transcriptase ! e.g., HIV!
Major drug target for AIDS since unique to virus !

*!

Latent phase; virus can hide for a long time!

Lytic phase !