Eukaryotic replication Flashcards
List and explain the cell cycle phases
The cell cycle is divided into 4 distinct phases:
> M phase (for mitosis)- mitosis and cell division occur during the relatively brief phase.
> G1 phase(for gap)- covers the longest part of the cell cycle, it is the main period for growth. Commitment to DNA replication.
> S phase (for synthesis)- which in contrast to to events in prokaryotes, is the only period in the cell cycle when DNA is synthesized. DNA replication.
> G2 phase- its relatively short. The now tetraploid cell prepares for mitosis. It then enters M phase again and the cell cycle starts again.
Discuss the variation that can occur within the cell cycle
The cell cycle for cells in culture is typically 16-24 hours, while cell cycle times for the different types of cells of a multicellular organism varies from 8 hours to >100 days. Most of this variation occurs in the G1 phase. Many terminally differentiated cells like neurons and muscle cells never divide so they assume a state known as the G0 phase. A cell’s irreversible decision to replicate is made in the G1 phase. A cell will remain in the G0 phase for e.g. if nutrients are in short supply.
Explain the control of the cell cycle
The cell cycle is regulated by proteins known as cyclins and cyclin-dependent protein kinases. In order to enter a new phase in the cell cycle, a cell must satisfy a checkpoint, which monitors if the cell has properly completed the preceding phase.
Briefly explain the DNA polymerases that occur in eukaryotic cells
The many known DNA polymerases can be classified into 6 families based on phylogenetic relationships (evolutionary relatedness). Members of families A,B and C are all replicative polymerases and some repair polymerases, family D occurs only in archaea and families X and Y participate in DNA repair. Animal cells express 4 distinct types of DNA polymerases- DNA polymerase alpha, gamma, sigma and E. Pol gamma is part of family A and occurs in the mitochondrion while the others are a part of family B and occur in the nucleus.
Describe DNA polymerase alpha
It occurs in the cell nucleus where it participates in the replication of chromosomes. Pol alpha replicates by extending a primer in the 5’-3’ direction under the direction of a ssDNA template. This heterotrimer, which lacks exonuclease activity, consists of a polymerase subunit, a primase subunit, a subunit that is required for full primase activity and a subunit for the regulation of initiation- all of which are collectively referred to as pol alpha/primase.
Describe DNA polymerase sigma
It is a heterotrimer whose catalytic subunit lacks an associated primase, but it contains a proofreading 3’-5’ exonuclease domain. Pol sigma exhibits unlimited processivity- it replicates the entire length of the template, in contrast to Pol alpha which exhibits moderate processivity- replicates about 100 nucleotides. Pol sigma can only replicate when it is in complex with PCNA (proliferating cell nuclear antigen).. PCNA is a trimeric ring with an almost identical structure as E.colis sliding clamp.
Describe the process of polymerase switching
Pol sigma in complex with PCNA is required for lagging strand synthesis. Pol alpha/primase synthesizes 12nt RNA primers and extends it with 20-30 nt of DNA. The next step involves polymerase switching. In polymerase switching, on the lagging strand replication factor C(RFC- like the E.coli clamp loader) displaces pol alpha and loads PCNA on the template DNA near the primer stand. Pol sigma then binds to PCNA and extends the DNA strand. On the leading strand pol alpha/primase is replaced by pol E, which is a highly processive polymerase that does not require PCNA to replicate. Pol E also has 3’-5’ exonuclease activity, but unlike pol E that excises mononucleotides at a time , it excises 6-7 residue oligonucleotides at a time
Discuss the replication origins in eukaryotes
Eukaryotic chromosomes have multiple replication origins, unlike prokaryotic chromosomes which have 1. Chromosomes do not replicate simultaneously, but rather in clusters as replicons (replication unit, DNA segments that are each served by a replication origin) that are activated simultaneously. A cell’s chromosomal DNA is replicated once per cell cycle.
Explain the assembly of the eukaryotic initiation complex
The assembly of the eukaryotic initiation complex occurs in two phases- a pre-replicative complex (pre-RC) is assembled at each replication origin during the G1 phase of the cell cycle- this process is called licensing. A licensed pre-RC cannot initiate DNA replication it must be activated to do so, this process occurs in the S phase (second phase of assembly). The assembly of the pre-RC and activation are separate- this ensures that a pre-RC cannot assemble at a replication origin that has already been used to initiate replication. The assembly of the pre-RC begins late in M phase or early in G1 phase with the binding of the origin recognition complex (ORC is similar to the DnaA in Ecoli) to the origin. ORC recruits two proteins- Cdc6 and Cdt1 which help load the MCM complex onto the DNA to yield the licensed pre-RC. MCM complex is similar to DnaB helicase and the two proteins are similar to DnaC(helps load the DnaB). The conversion of a licenced pre-RC to an active initiation complex requires the addition of pol alpha/primase, pol E, PCNA and Replicative protein A(like SSB)- this only occurs in the S phase.
How is re-replication prevented in eukaryotes
Once initiation (priming) has occurred, the initiation complex is joined by RFC and pol sigma and is converted to an active replicative complex by polymerase switching. DNA replication then continues bidirectionally until the replication forks collide, thereby completing the replication of the replicon (no ter sites or tus proteins in eukaryotes). An active replication fork will destroy any licensed pre-RCs and initiation complexes that have not been activated, this prevents replication from occurring twice. There are several mechanisms that ensure that a pre-RC can only initiate replication once- Cdks(cyclin-dependent kinases) are active from the late G1 phase through the late M phase, its required to activate initiation but also to prevent re-initiation. A protein called genminin appear in the S phase and continues to accumulate till the late M phase. Genminin associated with Cdt1 so as to inhibit the assembly of the pre-RC. It also protects the DNA against re-replication.
Explain how primers are removed in eukaryotes
Eukaryotes do not have pol capable of nick translation. In lagging strand synthesis, when pol sigma reaches the previously synthesized Okazaki fragment it partially displaces its primer, which generates an RNA flap. The primer is then removed by through the actions of 2 enzymes: RNase H1 removes most of the primer leaving only a 5’-ribonucleotide adjacent to the DNA, which is then removed by flap ensonuclease-1 (FEN1). Pol alpha/primase extends the RNA primers that it makes by 20-30 nucleotides before it is displaced by pol sigma, since it lacks proofreading ability it is more prone to contain errors. However, FEN1 is an endonuclease that excises mismatched bases and then the excised segment is replaced by pol sigma and the nick is sealed by DNA ligase.
Describe reverse transcriptase
The retroviruses, such as certain tumor viruses and human immunodeficiency virus (HIV) contain an RNA-directed DNA polymerase called reverse transcriptase. This enzyme synthesizes DNA in the 5’-3’ direction from an RNA template. Reverse transcriptase transcribes the retroviruses ssRNA genome to dsDNA: the retroviral RNA acts as a template for the synthesis of its complementary DNA, yielding a RNA-DNA hybrid helix. The RNA strand is then degraded by RNase H activity .The ssDNA now acts as a template for the synthesis of its complementary DNA, yielding dsDNA. During viral infection, the DNA is then integrated into a host cell chromosome. Reverse transcriptase is a useful tool in genetic engineering because it can transcribe RNA to cDNA. In transcribing eukaryotic mRNAs which have poly(A) tails, the primer can be oligo(dT).
Describe HIV-1 reverse transcriptase
HIV-1 RT is a dimeric protein that contains 2 subunits that are synthesized as identical 66 kD polypeptides, known as p66. p66 contains a polymerase domain and an RNase domain H. However the RNase H domain in one of the subunits is excised which yields a 51kD polypeptide called p51. Therefore HIV-1 RT is a dimer of p66 and p51. The 1st drugs to be clinically approved to treat AIDS include AZT which are RT inhibitors. Unfortunately, resistant strains of HIV-1 arise rapidly because they lack proofreading ability and are therefore error prone. Effective long term anti-HIV therapy requires the administration of at least one RT inhibitor and an HIV protease inhibitor.
Why can’t the ends of linear chromosomes be replicated by any of the mechanisms that we have considered so far
DNA polymerases require a free 3’OH group in order to extend DNA and this is usually provided by RNA primers. But, the RNA primer at the 5’-end of the lagging strand cannot be replaced with DNA because it does not have a free 3’OH group. It can be removed but cannot be replaced. This would result in the ends of chromosomes shortening after each round of replication, but the ends of chromosomes consists of telomeres.
Explain how telomeric DNA is synthesized
Telomeric DNA has an unusual sequence- it consists of up to several thousand tandem repeats of a simple, species-dependent G-rich sequence that ends the 3’-ending strand on each chromosome. The enzyme that synthesizes this strand is called telomerase. Telomerase is a ribonucleoprotein whose RNA component contains a segment that is complementary to a telomeric sequence. This sequence acts as a template in a kind of reverse transcriptase reaction that synthesizes the telomeric sequence, translocates to the DNAs new 3’-end and repeats the process. The 5’-end is then extended with lagging strand synthesis and it generates a 3’ overhang of the G-rich strand. The ends of a chromosome still shorten.
