Exam 2: Lecture 2 Flashcards Preview

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Flashcards in Exam 2: Lecture 2 Deck (19):
1

Initiation of DNA Replication (enzyme?)

-initial step of synthesizing new DNA strands is carried out by an enzyme called DNA primase
-enzyme will bind in a non sequence-specific manner to single stranded DNA and will generate a short (~10bp) oligonucleotide primer before it falls off the template strand
-can be made of DNA or RNA nucleotides
-leading strand will have fewer primers than the lagging strand
-All DNA/RNA oligonucleotide primers will be removed during one of the last steps of the replication process

2

DNA Primase

-highly error prone, which means that it adds the incorrect base quite frequently
-adds these bases very slowly
-a non-processive enzyme which means that it falls off the template strand (thus the primers are very short).

3

Bulk of DNA Replication

-carried out by DNA polymerase
-cannot bind to template DNA
-synthesize new DNA strands de novo therefore it locates the primer:template duplex instead and starts adding new bases that are complementary to the template strand

4

Polymerase MIstakes and Consequences

-has proofreading activity which means that most errors made by the polymerase are self-corrected
-speed at which the polymerase is adding/checking bases, a few mistakes are not self-corrected
-mutations fall into two broad categories – transition mutations and transversion mutations
-if polymerase induced mistakes are not corrected then they will be permanently encoded into the genomes of daughter cells
-can lead to disruptions of critical genes and will result in the onset of disease (and potentially its transmission to the next generation)
-surveillance systems within the nucleus that will correct any mistakes that escape the proofreading capabilities of the polymerase

5

Transition Mutation

-occurs when the wrong purine is placed opposite a pyrimidine or when the wrong pyrimidine is placed across a purine
-both cases the purine base pairs with a pyrimidine but the wrong match is made
-i.e. adding a G to a T instead of adding an A

6

Transversion Mutation

-occurs when a pyrimidine is incorrectly added to the new strand in the place of a purine or vice versa
-results in incorrect bases pairing between two purine or two pyrimidine bases
-i.e. adding a C to a T.

7

Polymerase is Processive

-kept from disassociating from the template strand by a multimeric protein complex called the sliding clamp
-interactions between sliding clamp and DNA polymerase are stronger that those between polymerase and template strand
-sliding clamp – polymerase interaction is what makes DNA polymerase a processive enzyme
-without sliding clamp, DNA polymerase can add only 10-14 bases before falling off template strand
-presence of slide clamp allows for single polymerase molecule to add several thousand bases at a time
-Mutations in the gene that encodes the human sliding clamp (PCNA) lead to S phase arrest (incomplete DNA replication

8

DNA Primase vs. DNA Polymerase

-primase creates a oligonucleotide primer that is ~10 bases in length because it cannot interact physically with sliding clamp, so it falls off after ~10 bases-->non-processive due to small number of bases it adds
-the DNA polymerase molecule, by interacting with the sliding clamp, can extend the primer by thousands of bases
-DNA polymerase is extending one primer to the next (filling gap between two primers)
-oncethe clamp-polymerase complex comes in contact with the next primer, the complex disassociates from the DNA template

9

Sliding Clamp Loader

-multi-subunit protein complex that plays a critical role in the process of replication
-simultaneously attached to two DNA polymerase molecules which are often oriented in opposing directions due to fact that synthesis of leading and lagging strand shappens in oposite directions
-each polymerase molecule is attached to its own sliding clamp and one of the two template strands
-sliding clamp loader bound to spare sliding clamp
-if either of the two polymerase-clamp complexes is disassociated from the template strand the sliding clamp loader will thread the template strands through the spare sliding clamp
-SCL assembles polymerase-clamp-DNA complex at the beginning of replication
-also aid in the dissociation of the entire complex when replication is complete

10

Leading and Lagging strands

-DNA polymerase reads the template strands in the 3`-5` direction and synthesizes the new strands in the 5`-3` direction
-the template strands are aligned in opposite orientations DNA synthesis of the two new strands occurs in opposite directions
-One of the two strands is synthesized in the same direction that the replication fork is moving-->leading strand, made in long continous stretches with few primer sequences (can be made in long stretches since polymerase is following helicase)
-other newly synthesized strand, called the lagging strand, is made in short discontinuous segments called Okazaki fragments and contains many more primers (primase and polymerase must constantly wait for new regions of the double helix to be opened up and made accessible)

11

Finishing Replication-RNase H

-removes oligonucleotide primers must be removed by an enzyme called RNase H
-belongs to a class of proteins called endonucleases – proteins that digest/eliminate nucleic acid
-will remove all but one of the primer nucleotides

12

Finishing Replication-Exonuclease

-removes last base of primer
-removes nucleotides that are internal to a long DNA duplex
-can remove nucleotides from the end of a DNA fragment or from a gap
-will be thousands of gaps along a chromosome. and each one has to be filled

13

Finishing Replication-Filling Gaps

-each gap is filled by DNA polymerase after last base of primer is removed, which now uses the adjacent DNA as a primer
-enzyme is unable to form a phosphodiester bond between the last base of the gap filled segment and the adjacent DNA
-ifthis phosphodiester bond is not made then that polynucleotide chain will fall apart during the next replication round or during transcription
-also there are nucleases that can detect these “nicks” in the phosphodiester backbone and could start to digest the genomic DNA

14

Finishing Replication-DNA Ligase

-a molecular glue
-recruited to make the last phosphodiester bond

15

Mutations Affecting Ligase

-mutations in the gene that encodes for fumaryllacetoacetate hydrolase lead to an accumulation of succinylacetone (SA), a precursor of the amino acid tyrosine
-accumulation of SA inhibits the activity of DNA ligase
-reductions in the activity of this key enzyme lead to a high level of chromosomal breakage due to the reduced joining of Okazaki fragments

16

Problem with Ends of Linear Chromosomes in Replication

-problem for replication machinery
-without special enzymes the lagging strand will be become shorter after each round of replication
-DNA polymerase can't fill in the very end of the chromosome after the last primer has been digested by RNase H (polymerase needs primer to function)

17

DNA Telomerase (Function and Composition)

-special polymerase that can extend the lagging strand
-composed of protein subunits and RNA species which contains a sequence that is found at the ends of chromosomes

18

DNA Telomerase (Sequence)

-binds to lagging strand then RNA will extend past the end of the lagging strand and will serve as a template
-DNA telomerase then adds bases to end of lagging strand that are complementary to RNA found within itself
-repeated several times and extends the overall length of chromosome

19

Reduction in Telomerase Activity

-implicated in aging process
-as ends of chromosomes (telomeres) get shorter gene coding regions begin to be eliminated leading to an assortment of diseases
-some studies show that increased DNA telomerase activity may increase lifespan