Hi again!
Let us begin by summarising what we learnt in the previous lecture on meiosis.
So, meiosis is reductional division as we now know and it is essential in order to maintain
genetic variation across the progeny and it is also essential to maintain the ploidy in
offspring.
There are different stages in the meiotic process starting with the G1 phase during
which the cells prep to enter meiosis.
During the S phase the DNA replicates, which is followed by the G2 phase.
G2 phase is followed by meiosis 1, which is an extremely important and it is where the
reduction of the number of chromosomes actually happens.
During prophase 1 in meiosis 1, which is subdivided into five different stages, in the pachytene
stage the homologous chromosomes to form the synaptonemal complex.
And during the pachytene stage crossing over or homologous recombination occurs.
During diplotene stage the synaptonemal complex disassembles, which is followed by anaphase
1 where the homologous chromosome segregation occurs and the number of chromosomes is reduced
to half.
After cytokinesis 1, meiosis 2 follows, which is again subdivided into prophase, metaphase,
anaphase and telophase.
What is noteworthy here is, during the anaphase two now sister chromatid segregation occurs
while the number of chromosomes stays the same.
Another very important point that is noted in the previous lecture was that in case there
is premature segregation of the homologous chromosome, which means that either both the
homologous chromosomes move towards the same pole and they do not move towards the different
opposite poles as they should have been, and in cases where the sister chromatid segregation
is premature or aberrant, the daughter cells that are formed will either be aneuploid or
polyploid; in both the conditions it is deleterious to the cells and can lead to fertility issues
and can also lead to you know genetic disorders in progeny.
We left at the question where we asked that even though the siblings have the same parents
why are they different from each other?
And how does the exchange of genetic material occur between the two homologous chromosomes.
And the answer to these questions lies in a process which is called homologous combination
and we will get into the details of how homologous recombination occurs.
And why is it very important during meiosis.
Now the homologous recombination as we know happens during the pachytene stage during
prophase 1 in meiosis 1.
However, other than that when cells are mitotically dividing homologous recombination is a process
which is essential to allow DNA repair.
However, in contrast to during meiosis, the preferred template is the sister chromatid
in case of mitotically dividing cells.
When homologous recombination occurs in context of meiosis, it is essential for the pairing
of the homologs first.
And the second thing is essential for the exchange of genetic material between homologs.
Now in contrast to mitotic dividing cells during meiosis the preferred template for
homologous recombination is the homologous non sister chromatids.
Even though even in the case of meiosis the sister chromatid is constitutively present,
some proteins actively make sure that the bias of using the preferred template is towards
non sister chromatid rather than the sister chromatid.
Now mechanistically homologous recombination can be divided into four different phases.
Let us that start with the first phase which is called the initiation phase.
During the initiation phase DNA double-strand breaks are induced in one of the homolog in
the pair by a specialised protein which we will discuss soon.
Now, you would wonder that why are we calling it as programmed double-strand breaks.
We are calling them programmed because this is one of the very few cases where the cell
is inducing the DNA damage in form of double-strand breaks.
And how you would wonder that in case there are a certain number of DNA double-strand
breaks in the cell maybe it will be deleterious to the cells and you are right, which is why
the number of DNA double-strand breaks during meiosis is highly regulated, as we will see
soon.
The initiation process is followed by the second step which is called the DNA double-strand
break processing wherein single stranded DNA tails are generated by a specialised set of
proteins and these single stranded DNA tails are recombinogenic in nature.
The second step is followed by the third step where the DNA is repaired using homolog as
we just saw, there is a bias towards the homolog.
And this process is followed by homologous recombination intermediate resolution and
all of these four processes involve a set of proteins which are specialised to generate
the homologous recombination intermediates, which are then resolved to give products,
which are called crossovers or non-crossovers.
On the left side we are showing a pair of homologous chromosomes one in green and one
in blue.
On the right side we are showing the stage wherein each of these mechanisms occur.
Now let us follow the schematic.
The first protein, which comes into picture during the initiation phase is called Spo11.
Now Spol11 is a topoisomerase like protein, which is evolutionarily conserved across organisms.
And what it does is, it induces the double-strand breaks in one of the homolog in the pair,
as you can see here the double-strand break is induced on the green homolog in the pair.
Following the DNA double-strand break induced by Spol11, there is another set of proteins
which is called Sae2 and Com1.
Now Sae2 and Com1 actively inhibit another form of DNA repair mechanism, which is called
nonhomologous end joining such that now the repair of this damaged DNA or the DNA wherein
the double strand breaks has been induced, occurs only using the homologous recombination
mechanism rather than using the nonhomologous end joining mechanism.
After the DSB’s, that is the DNA double-strand breaks, are induced, another set of proteins
which is called the Mre11 protein which has a 5 prime to 3 prime endonuclease activity
cleaves the DNA from the 5 prime end and this is called resection, which means that the
DNA is cleaved from the 5 prime end.
Similarly on this strand, which leaves an overhang in the 3 prime end and these single-stranded
tales are generated on the 3 prime ends.
And these single-stranded DNA tails are recombinogenic in nature and they are coated by another protein
called Rad51 which leads to the formation of a nucleoprotein filament here.
What is important to note here is that in most of the organisms the generation of the
DNA double-strand break is a prerequisite for homolog paring to occur and as you have
seen in the previous lecture the homolog start to co-align during the Leptotene stage.
So the DNA double-strand breaks are generated during the Leptotene stage.
The single stranded DNA tails on the three prime ends that have been generated here are
coated by Rad51 which forms nucleoprotein filament here.
This is absolutely essential because this is what is required for homology search.
And after the nucleoprotein filament is formed, it leads to homology search wherein it invades
the homologous template on the nearby homolog in the pair.
And this kind of structure that is found here is called a D-loop structure.
This intermediate that is formed during the process can now undergo one of the two pathways
wherein one of the pathways is called the crossover pathway which will generate crossover
products and the other pathway is called the non-crossover pathway which is which will
generate the non-crossover products.
This entire process is facilitated by different proteins namely Rad51, Dmc1, both of them
coat the single stranded DNA that is formed here.
Sae3, Rec8, Hop2 and Mnd1 are accessory proteins, which allowed the formation of the D-loop
intermediate here.
During the zygotene stage as we know earlier the proximity between the two homologs strengthened
furthermore by co-alignment.
Let us see mechanistically how that happens?
We ended up at a D-loop intermediate from where there are two pathways that the intermediate
can follow.
The first pathway is non-crossover pathway.
In this case the single stranded DNA end which extends itself or invades the homologous template.
DNA synthesis is primed on this template and a certain length of DNA synthesized.
After this, this extended end is extruded and this sort of intermediate is formed.
On the contrary, in the crossover pathway, there are several proteins namely Zip1, Zip2,
Zip3, Mer3 and Msh5.
These proteins facilitate single end invasion.
So, what happens is, as we see here the single stranded DNA invades the homologous template,
DNA synthesis is similarly primed.
However now in this case, another sort of intermediate, which is called as double Holliday
junction will be formed, which is not formed in the non-crossover pathway.
During the pachytene stage we know that the synaptonemal complex formation is complete,
which means that the two homologs are in such close proximity to each other that the distance
between them is almost equal to 100 nanometres, which means that the formation of these intermediates
is very intimate.
In case of the non-crossover pathway, the extruded end anneals to the primary homolog
and the DNA synthesis continues.
Now as the DNA is synthesized as you see in the white dotted line here, this entire homolog
is repaired and these products are formed which are called non-crossover products.
However, in the crossover pathway when the single end innovation occurs in DNA synthesis
occurs another homolog forms the second end annealing, the DNA synthesis continues and
ligation occurs as you can see here in white colour dotted line.
And an intermediate which is characteristic to the crossover pathway is formed, which
is called a double Holliday junction.
Now there are very important proteins, which are responsible in resolving this kind of
structure because DNA cannot be left in this kind of a mesh.
Therefore, specialised proteins called Holliday junction resolvases act on the cross structures
that are formed here and resolve the Holliday junction into the products that are called
the crossover products.
This process is completed during the diplotene stage of meiosis 1.
Note that per chromosome the meiotic machinery makes sure that there is one crossover.
And this one crossover per chromosome is called obligate crossover.
The obligate crossover is absolutely essential in order for the homologous chromosomes to
correctly segregate.
Also note that the number of DNA double-strand breaks that are generated by Spol11 during
the initiation phase is much, much larger than the number of crossover products that
are generated at the end, which means that not all DNA double-strand breaks end in the
formation of crossover products.
How the selection process occurs is as yet a matter of investigation and researchers
are actively looking into it.
Now let us discuss the significance of homologous recombination during meiotic prophase.
The exchange of genetic material between homologs is the primary reason for genetic variation.
And genetic variation in almost all of the organisms prove to be the basis for better
survival of offspring.
The next very important aspect is the proper segregation of chromosomes.
So, in many organisms, if the homologous recombination is impaired or the homologous recombination
does not occur, it adversely affects the segregation of chromosomes in a way that nondisjunction
occurs.
Now nondisjunction can lead to aneuploidy or polyploidy or genetic disorders such as
Turner syndrome, Down syndrome and Klinefelter syndrome.
The third very important aspect is homologous recombination in the canonical meiotic program
leads to homolog pairing.
In Saccharomyces cerevisiae, S. pombe, mammals, plants and filamentous fungi, homologous recombination
is absolutely essential to facilitate the homolog pairing.
However, this is a general rule, but there are alternative programs.
For example, in fission yeast and Asperigillus nidulans, what happens is both recombination
independent and recombination mediated pairing occurs, but the synaptonemal complex is absent.
In female Drosophila and C. elegans, the pairing between the homologous is independent of recombination.
In male Drosophila, recombination is absent and the pairing between the homologs is recombination
independent.
We shall see how the recombination independent paring occurs in a short while.
We just saw that DNA double-strand breaks lead to the formation of intermediates, which
facilitate homolog pairing and this is the canonical meiotic program.
However homologous pairing is also facilitated by interactions which are independent of DNA
double-strand breaks.
And you will see how the centromeres and the telomeres of the chromosomes are involved
in this process.
In cases of yeast and in mouse spermatocytes as you see here, centromeric associations
are formed long before synapses has occurred in the prophase stage.
As you can see the homologous chromosomes in green and purple are shown here.
Now, there are two events which are called centromeric coupling and centromeric pairing.
Centromeric coupling usually occurs between homologous and can also occur between nonhomologous
chromosomes.
However, centromeric pairing occurs only between homologous chromosomes.
In yeast it has been shown that is Zip1 protein is essential for both centromeric coupling
and centromeric pairing and the localisation of Zip1 to the centromeres is facilitated
by another protein, which is called Rec8.
After centromeric coupling has occurred during the prophase synapsis occurs and the homologous
chromosomes pair together, which is called centromeric paring.
And then the cell cycle proceeds towards metaphase.
However, in case of mouse spermatocytes centromeric coupling has not been detected.
Synapsis occurs and is followed by centromeric pairing which is followed by metaphase.
So what we see here is, centromeres play an important role in homolog pairing even before
double-strand breaks are generated.
Therefore, this is one of the mechanisms wherein homolog pairing occurs independent of the
DNA double-strand breaks.
Now you would wonder why this kind of process occurs?
Now inside a cell where one homolog has to find the other homolog in order to pair together,
the centromeric coupling allows them to check transient interactions and in case they find
the correct homolog this interaction is stabilized during synapsis and is matured into pairing.
So therefore, these transient interactions that occur long before actual synapsis has
occurred, make sure that the pairing process is made easier and the finding of the homologous
is made, correct.
Another way by which homolog paring occurs independently of the DNA double-strand break
generation is by exploiting the telomeres of the chromosomes.
Now in early Leptotene all the telomeres tether to the nuclear envelope, which is shown here
in the blue boundary.
During the late Leptotene, the telomeres start moving towards one of the ends of the nuclear
envelop.
This clustering of telomeers is completed during the zygotene stage as you see here.
And during the pachytene stage the scattering of telomeres starts.
Now in plants and vertebrates, telomeric interaction leads to formation of a bouquet structure
also called the REBL orientation.
The telomeres tethered to the nuclear envelope.
In fission yeast it has been shown that mutations that affect the telomeric clustering lead
to reduced recombination.
And such mutations are used to study the effect of the telomere clustering on homolog pairing
and subsequently on homologous recombination.
Experiments in Saccharomyces cerevisiae have shown that homolog recognition requires telomeres
and telomere associated protein called Ndj1.
Now mutations in Ndj1 show impaired chromosome pairing and bouquet formation.
Except C. elegans and Drosophila bouquet formation is found in all studied organisms.
Other than the role of telomeres and centromeres in homolog pairing during double-strand break
independent homolog pairing, there is another candidate, which is absolutely essential in
order to allow homolog pairing and these are called the non-coding RNA’s.
Studies in fission yeast show that Mei2 encodes an RNA binding protein which functions at
two stages.
Primarily, Mei2-RNA complex sequesters a negative regulator of meiosis called Mmi1, which binds
meiotic specific transcripts during mitotically dividing cells and prevents meiosis in mitotically
dividing cells.
During meiosis however the Mei2 RNA accumulates at SME2 locus and they promote homolog recognition
and pairing of homologous chromosomes.
Let us now look at the summary of the lecture of part 2.
We started with homologous recombination.
We now know that during meiosis inter homolog recombination is preferred over inter-sister
recombination, which is preferable during mitosis when the cells are divide mitotically.
We also studied how homologous recombination occurs.
It starts with the introduction of DNA double-strand breaks by a topoisomerase like protein called
Spol11.
Mre11 and other endonucleases generate single strand tails which are recombinogenic in nature,
they are bound by proteins called Rad51, that nucleoprotein filament is now ready for strand
invasion.
After strand invasion intermediates are formed typically during crossover process.
The double Holliday junction is formed; proteins called Holliday junction resolvases resolve
such intermediates.
And then there is the generation of crossover and non-crossover products.
We also studied that homologous recombination is responsible for homolog pairing in the
canonical meiotic pathway.
It is essential for proper chromosome segregation and it is absolutely essential for genetic
variation.
Next, we studied that homolog pairing is not just double strand break dependent it is also
independent of the DNA double-strand breaks.
It can be mediated either by centromeres.
It can also be mediated by telomeres and can also be mediated by small RNAs.