Textbook 1.2 Flashcards
Pg. 394-398, 382-387, 460-461, 465-470, 480-482, 512-526
DNA Gyrase
Type of topoisomerase that relieves mechanical strain that builds up during replication in E. coli (super coiling). Cleaves DNA duplex in two and passes one of the strands through. Eukaryotes have similar enzymes which have same function.
For DNA polymerase to work…. must be present
DNA and all four deoxyribonucleoside triphosphates dTTP, dATP, dGTP, dCTP
All DNA polymerases CANNOT…and thus require …
- initiate the formation of a new DNA strand—can only add nucleotides onto the 3’ OH terminus of an existing strand
- a template strand to copy and a primer to provide the OH end
What is the major enzyme responsible for DNA replication…
DNA Polymerase III, even though there is far fewer copies of the enzyme than Polymerase I which was discovered first.
Replication is said to be…
semi-discontinuous
Which enzyme links Okazaki fragments together
DNA ligase
Which enzyme initiates DNA replication
An RNA polymerase called RNA primase which constructs a short primer of RNA
What happens to the RNA primers at the end of replication?
They are removed and replaced with DNA. The strands are then sealed by DNA ligase.
What is a possible reason for an RNA primer being used?
Potentially more mistakes made during initiation than elongation so a short, removable RNA primer avoids inclusion of mismatched bases.
Helicase
Unwinding enzyme that uses energy from hydrolysis of ATP to move along strand. Forms a ring like structure around strand.
SSB Proteins
Single-stranded DNA-binding proteins: bind selectively to single stranded DNA to keep it extended and prevent it from rewinding or getting damaged.
Primosome
Transient association between primase and helicase in bacteria during replication.
Describe the polymerase complex during replication
Both the leading and lagging strand have separate DNA polymerases which are adding onto the 3’ OH terminus of the leading and lagging strand. The DNA polymerase on the leading strand is synthesized continuously while the protein on the lagging strand synthesizes in chunks then releases template to move along to the next fragment. It is hitching a ride on the other polymerase. They are part of one complex even though there are two polymerases because the DNA of the lagging strand template is looped back on itself. They are moving towards opposite ends of their respective templates but ultimately still in same direction.
DNA polymerase III holoenzyme
replication machine. Includes:
- DNA polymerase III
- beta clamp (keeps polymerase associated w template)
- clamp loader (holds clamp open for DNA to squeeze into ring of beta clamp)
Difference between beta clamp and DNA polymerase III association on the leading and lagging strand
Polymerase on the leading strand remains tethered to the beta clamp for the entire duration of the replication while polymerase on the lagging strand disengages from the beta clamp after completing an Okazaki fragment and is cycled to a new beta clamp on an RNA primer-DNA template junction closer to replication fork.
Exonucleases can be divided into…
5’ to 3’ and 3’ to 5’ depending on the direction of the degradation of the DNA strand
Why is DNA polymerase I almost like three enzymes in one?
It has polymerizing capabilities but also 3’ -> 5’ and 5’ -> 3’ exonuclease capabilities. These activities are found in three different domains of the polypeptides. The 5 to 3 exonuclease is not RNA/DNA specific unlike most other exonucleases and can degrade both.
Which enzyme removes the RNA primers that initiate okazaki fragments?
5’ to 3 ‘ exonuclease activity of DNA polymerase I. It also fills the gaps as it removes the primers.
Which enzyme seals okazaki fragments together
DNA ligase
How does DNA polymerase ensure the right nucleotides are being added?
Bond angles and geometry of base pairs. If the nucleotide has the proper fit the enzyme will undergo a conformational change to add onto the growing chain. If it does not have proper geometry enzyme cannot achieve conformation required for catalysis.
What happens if the wrong nucleotide is added?
The frayed end of the 3’ strand is fed into the 3’ to 5’ active nuclease site (at wrist) where the incorrect base is removed. This removes 99 out of every 100 mismatched bases.
Fidelity of DNA replication relies on…
- accurate selection of nucleotides
- immediate proofreading
- post-replicative mismatch repair
Human genome contains about
21 000 protein coding genes, same number as microscopic worm whose entire body has only about 1000 cells.
90% of human genes participate in…
alternative splicing which allows for one gene to encode a number of related proteins.
What can explain the difference in complexity between organisms?
It’s less a result of amount of genetic information in an organism’s genome and more the manner with which it is put to use.
FOXP2
Difference in two amino acids between chimp and human version of this protein. Mutations of this gene cause severe speech impairment in humans. Possible speech gene?
SRGAP2
duplicated in humans, increases number of dendritic spines (may have contributed to our brain size) .
AMY1
encodes for starch digesting enzyme amylase. Humans have more copies on average (amount is variable).
Highly repeated DNA sequences
constitutes 1-10% of total DNA. Given sequence repeats itself over and over with no interruptions. Includes satellite DNA, mini-satellite DNA, and micro-satellite DNA.
What is mini-satellite DNA used for?
DNA fingerprinting and identifying individuals in criminal/ paternity cases. This DNA is highly variable from individual to individual.
Where is satellite DNA found?
It’s localized in the centromeric regions of chromosome
FISH
Fluorescence in situ hybridization
- utilizes satellite DNA and re-annealing capabilities to label DNA fragments
Moderately repeated DNA sequences
Can constitute 20-80% of total DNA depending on organism. Interspersed throughout genome. Does not occur in tandem sequences (repeats right after another).. Includes SINEs and LINEs.
Non repeated DNA sequences
localize to a particular region on a particular chromosome and includes DNA sequences that code for virtually all proteins other than histones.
DNA of prokaryotes is not…
packaged into chromatin, so DNA binding proteins can bind directly to preferred binding sites
Nuclear envelope
Biggest distinction between prokaryotes and eukaryotes. Forms boundary between nucleus and cytoplasm and consists of two cellular membranes which are fused at sites to form circular pores.
In the typical interphase (non-mitotic) cell, the nucleus contains…
chromosomes as chromatin, nucleoli (electron dense structures), and nucleoplasm
Nuclear Lamina
provides mechanical support to nuclear envelope, site of attachment for chromatin fibres at the nuclear periphery. Filamentous and meshy.
Heterochromatin does not extend…
into the region of the nuclear pore
Average human cell has…
6.4 billion bp divided among 46 chromosomes
The larger the chromosome
the longer the strand of DNA that makes it up
Chromatin
Stuff that makes up chromosomes—composed of DNA and associated proteins
Histones contain an unusual amount of
arginine and lysine
Histones
Responsible for orderly packing of eukaryotic DNA. There are five types: H1, H2A, H2B, H3, H4
Which of the histone proteins have undergone the least evolutionary change?
H3 and H4, though none of them have changed that much because the positive charge of this peptide is responsible for associating with the negative backbone of the DNA molecule (same in all organisms).
Nucelosomes
Contains a core particle consisting of 14 bp of supercoiled DNA wrapped almost twice around a histone octamer.
What’s important about the N-terminal segments of histones?
They are targets of key enzymes that play a key role in making the chromatin accessible to proteins.
Lowest level of chromatin structure
DNA molecule wrapped around nucleosome core particle of 10 nm diameter
What have linker histones and core histones been implicated with?
Higher-order packing of chromatin. Without H1, 30 nm fibres uncoil to form beaded filament. Chromatin fibres without H4 histone tails cannot fold into higher order fibres.
Cohesion
protein that maintains DNA loops when 30 nm fibre is coiled/looped up into thicker fibres
Heterochromatin
chromatin that remains compact during interphase (little transcriptional activity)
Euchromatin
dispersed, active state
Constitutive heterochromatin
Remains in compacted state in all cells at all times (silenced DNA), usually found in regions that flank the telomeres and at centromere. When active genes move to this region they tend to become transcriptionally silent. Inhibits genetic recombination between homologous repetitive sequences.
Facultative heterochromatin
Chromatin that has been specifically inactivated during certain stages of an organism’s life. Ex. only one of a female’s X chromosomes is transcriptionally active while the other is a heterochromatic clump (barr body).
X-chromosome inactivation
- Heterochromatinization of X chromosome occurs early in embryonic development
- Paternally and maternally derived X chromosome have an equal chance of being inactivated in any given cell (any descendent cell after has the same X chromosome inactivated)
- Reactivation of the Barr body occurs in female germ cells prior to onset of meiosis - both x chromosomes are active during oogenesis.
Adult females are
genetic mosaics; different alleles function in different cells
Where are inactive or less gene rich chromosomes found
periphery of nucleus
