Cover Letter

To: Prof. Kimberly Freeman, COLWRIT 161, College Writing Program

From: Fenmiao Zhong

Date: October 17, 2023

Subject: Formation and Repair of Meiotic Double-strand Breaks in Caenorhabditis elegans

My literature review discusses the formation and repair processes of double-strand breaks

(DSBs) in the nematode, Caenorhabditis elegans (C.elegans). C.elegans is a favorable genetic

model for studying steps in meiosis since there are well-established visualization and genetic

manipulation methods for experiments. The formation of double-strand breaks involves several

highly conserved proteins such as SPO-11, DSB-1, DSB-2, DSB-3, and less conserved proteins

such as HIM-17. They play different roles and interact with each other to collectively regulate

the DSB initiation process. DSB repair pathways include non-homologous end joining (NHEJ)

or homology-mediated repair, and each has its niche in monitoring and responding to DNA

damage signals. Misregulation of DSBs can lead to severe consequences such as chromosome

missegregation or non-disjunction and embryonic lethality, so understanding the proper

regulation is important for identifying targets for fertility problems or reproductive diseases.

Other protein and non-protein factors can directly or indirectly affect the DSB levels in

C.elegans germline, including synaptonemal complexes, cohesins, condensins, age, and sperm,

though exact pathways are still being investigated.

If I were to submit the review to a journal, I would submit it to Frontiers in Cell and

Developmental Biology, which is a broad-scope, interdisciplinary open-access journal, with a

focus on fundamental biological processes. The journal has specific sections such as Cellular

Biochemistry and Molecular and Cellular Reproduction that might fit the topic of this review, as
my review focuses on DSBs, a key cellular process on a molecular basis. I use the Author-Date

in-text citation and order the reference list in alphabetical order, which matches the review

articles published in this journal.

In reading this review, it may be helpful to know the following terms to understand the review:

● Chiasmata: It is the point of contact between homologous chromosomes and specifically

on non-sister chromatids, where genetic information can be exchanged between linked

chromatids via a process called crossover. Due to such physical contact, homologous

chromosomes pair up with each other and form bivalents consisting of two homologous

chromosomes in normal meiotic progression. Meiotic defects can result in a loss of such

physical linkage and formation of univalents since homologous chromosomes are not

brought together in proximity, which prevents crossovers.

● Immunostaining: It is a biochemical technique that uses antibodies to detect a specific

protein of interest. Specifically in C.elegans studies, immunofluorescence, one type of the

common immunostaining methods, is widely used. The technique involves applying

antibodies that target fluorescent dyes or fluorophores to the protein of interest in

C.elegans gonad/germline samples, so that the distribution of the targeted protein can be

visualized under fluorescence microscopy.

Formation and Repair of Meiotic Double-strand Breaks in Caenorhabditis elegans

Abstract

Proper regulation of meiotic double-strand breaks (DSBs) is fundamental for meiotic progression and

recombination. Misregulation in the formation and repair of DSBs can lead to meiotic defects and

chromosome abnormalities such as crossover (CO) failure, univalent formation, and chromosome

missegregation, impairing genome integrity. For meiosis studies, Caenorhabditis elegans (C.elegans) is

widely used as a model organism due to its simplicity, amenableness to genetic manipulation, and

transparency. To examine DSB levels, an important readout for meiosis progression, several visualization

methods such as immunostaining of proteins important in the DSB repair pathway and assessing bivalent

formation via DAPI staining as well as phenotypic readouts such as embryonic viability and frequency of

XO males in the population are utilized. This review summarizes key proteins mediating DSB formation

and repair, consequences of defective DSB regulation, and other factors, including chromosome structure,

reproductive age, and sperm signal, impacting DSB levels, in C.elegans meiosis. Though many

significant factors in meiotic recombination have been identified, many molecular details of their

interactions and regulation mechanisms are still unclear. Further characterization of protein regulators and

in-depth research are needed to extend people’s understanding of important stages in meiosis, which will

shed light on tackling fertility problems and maintaining genome integrity.

Keywords: double-strand break, crossover, C.elegans, meiotic recombination, genome integrity

Introduction

Meiosis is a crucial biological process in the life cycles of organisms undergoing sexual reproduction. It

consists of one round of DNA replication followed by two subsequent rounds of cell divisions, in which

homologous chromosomes and sister chromatids segregate respectively during the first and the second

cell division (Yu et al., 2016). To ensure proper chromosome segregation, homologous chromosomes

must recognize and pair with each other, establish synaptonemal complexes, and carry out crossover

recombination, resulting in the formation of chiasmata. Meiotic recombination events are initiated by

programmed DNA double-strand breaks (DSBs) during the first round of meiotic cell division. Those

DSBs are generated by an evolutionarily conserved topoisomerase-related endonuclease SPO-11. A

portion of programmed DSBs are repaired via the homologous recombination pathway and are resolved to

form crossovers (COs) between homologous chromosomes (Rosu et al., 2013). DSBs are crucial to

promote crossovers on every chromosome pair, but excessive DSBs can result in genome instability by

introducing mutations into the genome (Stamper et al., 2013). Thus, DSB formation and repair must be

highly regulated to balance the requirement for CO formation and genome integrity.

This review provides an overview of the formation and repair processes of DSBs in Caenorhabditis

elegans germline. As an established model organism for meiotic studies, C.elegans embrace many

favorable characteristics such as simple structure and genome, convenient genetic experimentation, and

transparent germline for visualization. Some robust experimental methods based on staining to examine

protein abundance and localization have been developed to study DSBs in meiosis. Many essential factors

involved in the process have been characterized using those methods in C.elegans models. In this review,

some key proteins involved in DSB formation and repair are introduced, followed by the consequences of

DSB defects. Other non-protein factors, such as chromosome structure, age, and sperm exposure, are also

discussed. Despite the relative simplicity of C.elegans, how all proteins interact with each other and

collectively regulate the level and distribution is not fully understood. Further research on the molecular
mechanistic details and functionality of those DSB-related proteins and indirectly relevant factors are

needed for a more comprehensive understanding of DSB and meiotic recombination, aiming towards

tackling complicated reproduction defects and fertility problems in a broader context.

C.elegans as a model system to study meiotic events

Caenorhabditis elegans (C.elegans) is a good model organism for the study of meiosis due to a variety of

reasons. C.elegans is a small organism that can easily grow in the laboratory setting with minimal

requirements of food and space. It has a short lifespan of about two to three weeks, allowing multiple

generations and meiosis over life cycles to be studied in a relatively short time course. C.elegans is simple

in terms of the number of cells and protein-coding genes, permitting tracking behaviors of individual cells

or functions of individual genes. C.elegans is also favorable for genetic manipulation through forward and

reverse genetic approaches (Garcia-Muse et al., 2007). It is easy to introduce mutations or transgenes via

gene editing tools such as CRISPR-Cas9 and silence genes via RNA interference (RNAi) to study specific

genes involved in meiosis. Despite its simplicity, C.elegans share essential meiotic genes and fundamental

molecular pathways with other complex organisms, which facilitates understanding meiotic progression

from an evolutionary perspective and applying knowledge to a broader context.

C.elegans’ transparent gonad enables visualization of chromosomal organization and protein localization

at different stages throughout meiotic progression (Garcia-Muse et al., 2007). In the adult C.elegans

hermaphrodite, germline nuclei enter meiotic prophase I after a short pre-meiotic region at the distal tip of

the gonad. The meiotic prophase I has five stages – leptotene, zygotene, pachytene, diplotene, and

diakinesis – associated with different chromosome morphologies and hallmarks. Initial homologous

chromosome pairing events occur in the leptotene-zygotene (transition zone) where chromosomes have

characteristic crescent shapes. In early pachytene, homolog synapsis as a result of synaptonemal complex

formation takes place and is complete by mid-pachytene. The chromosomes begin to de-synapse and
condense into chiasmata in the diplotene. In diakinesis, nuclei can be visualized as six distinct

DAPI-stained bivalents representing highly condensed homologous pairs. Understanding the events

happening during each stage will be important to unveil the molecular mechanisms and genetic regulation

involved in each stage.

Methods to examine DSB levels

In C.elegans, accurate chromosome segregation requires the establishment of the meiotic recombination

crossovers as a result of repaired meiotic DSBs via the homologous recombination (HR) pathway. Meiotic

DSB repair by HR pathway can be monitored by measuring crossover frequency based on phenotypic

markers or analyzing the presence and distribution of key proteins in the repair pathway (García-Muse,

2021).

The most common method is RAD-51 immunostaining. RAD-51 is a key protein involved in the HR

pathway. It is recruited to the single-stranded DNA overhangs at DSB sites generated by the endonuclease

SPO-11 to form nucleoprotein filaments and promote strand invasion as well as recombination between

homologous chromatids. Since RAD-51 specifically assembles at DSB sites, its presence designates the

programmed DSBs that are undergoing repair (García-Muse, 2021). During C.elegans meiosis, RAD-51

starts localizing to discrete foci in nuclei upon meiotic entry, becomes the most abundant at early

pachytene, and disappears after late pachytene (García-Muse, 2021). Immunostaining of RAD-51 in

C.elegans germline leads to the formation of distinct spots in the nuclei and provides information about

DSB formation and repair sites. Quantitative analysis of RAD-51 foci number and intensity allows

assessment of DSB levels in the germline.

Alternatively, RPA-1 immunostaining is used to study meiotic DSBs. RPA-1 binds to single-stranded

DNA at DSB sites to protect single-stranded overhangs and facilitate DSB processing, leading to the
formation of RPA foci which can be easily visualized upon immunostaining. Since RPA-1 binding to

single-stranded DNA after resection precedes RAD-51 binding, quantitative analysis of RPA-1 foci

number and intensity allows a more immediate understanding of DSB formation and repair process

(García-Muse, 2021).

COSA-1 immunostaining is used as a less direct indication for DSB levels. COSA-1 localizes to foci

corresponding to the single crossover (CO) site on each homologous chromosome pair during the late

meiotic prophase and promotes the conversion of DSBs into COs (Yokoo et al., 2012). Defects in DSB

formation can lead to a reduction in the number of COs formed. Due to the small number of COSA-1 foci

compared to that of RAD-51 or RPA foci in each nucleus, quantification of COSA-1 foci is less tedious

while providing sufficient information for estimating the extent of DSB formation and homologous

recombination repair.

Besides immunostaining of key proteins involved in the DSB formation and repair pathway, several

phenotypic markers are useful for assessing the success of meiotic recombination initiated by DSB

formation. During diakinesis, chromosomes condense and appear as discrete bodies. In the wild-type

C.elegans, 6 DAPI bodies that represent 6 homologous chromosome pairs, or bivalents, can be seen per

nucleus. An increase in the number of DAPI bodies observed suggests the formation of univalents,

resulting from failure in pairing and/or crossovers. The number and distribution of DAPI-stained bodies in

the diakinesis correlate with the formation of COs, which is a direct outcome of DSB repair. Changes in

the number of DAPI bodies imply variations in DSB formation and repair process, so counting DAPI

bodies serves as a complementary method to gain a more comprehensive view of DSB levels and meiotic

progression.

Oftentimes, a combination of methods described above is used in a study to provide a more

comprehensive examination of DSB levels from different aspects.

Proteins required for meiotic DSB formation

SPO-11

SPO-11 protein is a highly conserved topoisomerase-related protein present in all sequenced eukaryotic

genomes. SPO-11 cuts double-stranded DNA endonucleolytically and generates a covalent protein-DNA

intermediate by attaching to the DNA at the cleavage sites. After DSBs are formed, SPO-11 is removed

from DNA and 5’ ends are resected to produce 3’ single-stranded tails. The single-stranded tails undergo

strand invasion to promote the formation of recombinant products. DSBs repaired via different pathways

can give rise to either crossovers, with reciprocal exchange of genetic information, or non-crossovers

without such genetic exchange. In C.elegans, SPO-11 is primarily expressed in meiotic cells (Keeney,

2008). DSBs induced by irradiation can partially restore meiotic crossover formation in spo-11 mutants,

which implies that SPO-11 is essential for DNA scission activity that initiates meiotic DSB formation

(Dernburg et al., 1998).

HIM-17

In C.elegans, HIM-17 is recruited to germline chromosomes. Defective chromosome segregation and

chiasmata formation while undisrupted pairing and synapsis in him-17 mutants indicate defects in the

meiotic recombination process. Similar to spo-11 mutants, radiation-induced DSBs can rescue crossover

formation in him-17 mutants, reinforcing the important role of HIM-17 in mediating Spo11-dependent

DSB formation (Reddy et al., 2004). Besides, loss of HIM-17 results in a reduction of acquisition of

lysine 9 methylation of histone H3 (H3MeK9) on germline chromatin, suggesting that HIM-17 may

promote accumulation of H3MeK9 to regulate SPO-11 activity of DSB initiation (Reddy et al., 2004).

DSB-1, DSB-2, DSB-3

DSB-1 (Double-Strand Break factor 1) is required for meiotic DSB formation and localizes preferentially

to autosomes and less to X chromosomes, though it regulates DSBs on all chromosomes (Stamper et al.,

2013). C.elegans dsb-1 mutants show several meiotic defects, including decreased progeny viability,

higher incidence of male progeny, lack of DSBs, and failure of crossover formation, while homologous

chromosome pairing and synapsis remain undisturbed. Irradiation-induced exogenous DSBs can rescue

recombination defects, indicating that DSB-1 is an essential factor required for meiotic DSB formation

(Stamper et al., 2013).

DSB-2 (Double-Stand Break factor 2), a paralog of DSB-1, promotes meiotic DSB formation. C.elegans

dsb-2 mutants exhibit meiotic defects such as failure in crossover formation that worsen upon aging, but

they are proficient for homologous chromosome pairing and synaptonemal complex assembly, suggesting

a deficiency in meiotic recombination (Rosu et al., 2013). Irradiation also bypasses the need for DSB-2

and rescues bivalent formation, indicating that DSB-2 is specifically required for DSB formation.

DSB-1 and DSB-2 have similar distribution patterns in the germline but they do not colocalize

extensively (Stamper et al., 2013). Examination of DSB-1 and DSB-2 localization in the absence of each

other and comparison of mutant phenotypes reveal that DSB-1 and DSB-2 mutually facilitate each other’s

expression and stabilization to collectively regulate DSB formation, with a greater dependency of DSB-2

on DSB-1 (Stamper et al., 2013).

DSB-3 is a recently characterized DSB-promoting protein partnering with DSB-1 and DSB-2. Similarly,

loss of DSB-3 does not affect pairing and synapsis but results in fewer endogenous DSBs (Hinman et al.,

2021). DSB-3 signals are abolished in dsb-1 null mutants and are reduced in dsb-2 null mutants,

suggesting the interdependence of DSB-3 with DSB-1 and DSB-2. Furthermore, DSB-1, DSB-2, and

DSB-3 form a complex that is a distant functional analog of the Rec114-Mei4 complex, required for DSB

formation in meiotic recombination for fungi and mammals, based on Y2H interactions, colocalization,
and sequence and structural alignment data (Hinman et al., 2021). However, the exact molecular details of

interactions between those DSB-promoting factors and how the DSB-1-DSB-2-DSB-3 complex regulates

Spo-11 activity and facilitates DSB formation still need investigation.

DNA DSB repair pathways

In C.elegans, DSBs are repaired via conserved pathways, non-homologous end joining (NHEJ) or

homology-mediated repair. NHEJ is an error-prone pathway that joins broken DNA ends together without

requiring sequence homology, so mutations in the genome such as insertion and deletion (indels) are

likely to occur (Lemmens et al., 2011). Homology-mediated repair includes mutagenic repair pathways

single-strand annealing (SSA) and high-fidelity repair pathway homologous recombination (HR). SSA

utilizes sequence homology extending the DSB to anneal and ligate resection ends. HR uses a sister

chromatid or a homologous chromosome as a homologous DNA repair template to restore the sequence

information lost at the break sites (Lemmens et al., 2011).

Consequences of abnormal DSB formation and repair

DSB formation and repair pathways coordinate to safeguard genome integrity. Abnormal DSB formation

and faulty DSB repair in C.elegans germline leads to the accumulation of excessive DSBs. Depending on

the repair context, cell cycle stage, and DNA conditions, different types of developmental abnormalities

become obvious to different extents, and they are often used as readouts for meiotic defects and genome

instability (Lemmens et al., 2011). Common germline defect readouts include embryonic

viability/lethality, XO male frequency, and nuclear morphology in the diakinesis. A direct consequence of

misregulated DSB formation and repair is crossover defects. Failure to form crossover or excessive

interference with crossover formation can cause missegregation of homologous chromosomes during

meiosis, leading to autosomal non-disjunction and/or X chromosomal non-disjunction. The

non-disjunction can ultimately lead to aneuploidy, reducing embryo viability. Moreover, increased X
chromosome non-disjunction can contribute to a manifestation of males (XO) in the normally

hermaphrodite-dominated (XX) population, causing a high-incidence-of-males (HIM) phenotype.

Screening of HIM phenotypes is a robust method to reveal novel DSB formation and repair factors

essential to meiotic recombination events (Lemmens et al., 2011).

Chromosome structure and DSB formation and repair

Synapsis

Synaptonemal complexes (SCs) form between homologous chromosomes during the meiotic prophase,

regulating DSB formation and repair of DSBs to COs on homolog pairs (Hollingsworth, 2020). SC

formation starts with the condensation of sister chromatids to make axial elements, which are zipped

together by filaments perpendicularly and central elements parallelly. The axial element recruits proteins

such as SPO-11 that are required for DSBs and other proteins monitoring meiotic checkpoints. The central

region, comprised of filaments and central elements, downregulates SPO-11 activity (Hollingsworth,

2020). By removing DSB machinery upon the success of SC assembly, synapsis controls the proper

timing of DSBs to prevent excessive DNA damage.

Cohesin

In C.elegans, two meiotisis-specific cohesin complexes, REC-8 and COH-3/4, are expressed. They are

components of axial elements and have overlapping and non-overlapping functions in axis assembly,

sister chromatid cohesion, pairing, synapsis, and recombination (Castellano-Pozo et al., 2020). REC-8 and

COH-3/4 have been recently identified to play a role in promoting SC stability in pachytene nuclei, and

loss of either cohesin causes SC disassembly, which compromises DSB formation (Castellano-Pozo et al.,

2020). However, no direct role of cohesin affecting DSB formation and repair has been identified or

characterized and this still needs to be confirmed or supplemented by further studies.

Condensin

In C.elegans, there are three types of condensin complexes, Condensin I, Condensin II, and Condensin

IDC, each composed of two SMC proteins and three non-SMC proteins (Mets et al., 2009). The SMC

protein MIX-1 is shared by all three condensins, and the other SMC protein can be either SMC-4, as in

Condensin I and II, or DPY-27, as in Condensin IDC. Condensin I and IDC share all non-SMC proteins,

DPY26, DPY-28, and CAPG-1. Condensin II has a different set of non-SMC proteins, including KLE-2,

HCP-6, and CAPG-2. Among them, Condensin I is responsible for meiotic crossover control by

regulating the number and distribution of meiotic DSBs and COs (Mets et al., 2009). An increase in DSB

production as well as the following increase in CO formation in dpy-28 mutants suggests an important

role of condensin units in restricting DSB number and thereby CO number for meiotic progression (Mets

et al., 2009). Disruption of one or more Condensin I subunits extends the length of chromosome axis in

the pachytene, designating a role of Condensin I in regulating chromosomal structure. Since the

establishment of chromosome axis occurs prior to DSBs and is indispensable for DSB formation, DSB

frequency is likely to be directly mediated by chromosome axis length controlled by Condensin I (Mets et

al., 2009). How axis length is involved in the reshape of chromosome structure and how chromosome

structure determines DSB distribution and frequency are still worth exploring.

Other factors impacting DSB formation and repair

Reproductive Age

C.elegans hermaphrodites continuously generate oocytes after they mature to the adult stage and undergo

infertility worsening upon aging due to declined oocyte quality (Toraason et al., 2022). As genome

instability negatively affects oocyte quality, DSB formation and repair processes, which are crucial for

genome integrity maintenance, can be impacted by an increase in reproductive age, contributing to

age-related fertility problems. Recently, careful quantification of RAD-51 foci in different stages of

meiotic prophase in young and old worms shows that old germlines have reduced accumulation of
RAD-51 foci in the early pachytene and elevated levels of RAD-51 foci in the mid pachytene compared to

young germlines, indicating a reduced DSB induction and delayed DSB repair as worms age (Toraason et

al., 2022). Moreover, after irradiation, aged germlines exhibit impairment in recruitment, accumulation,

and removal of both RAD-51 and RPA-1 at DSB sites, suggesting severe defects specifically in the HR

repair pathway (Raices et al., 2021). More studies need to be conducted to understand how different repair

pathways are impacted by increased reproductive age distinctly and how aging regulates individual steps

in DSB formation and repair thus leading to oocyte quality deterioration.

Sperm Signal

Exposure to sperms or sperm-specific signals can affect levels of RAD-51 in C.elegans germline

(Toraason et al., 2022). Sperm depletion is shown to downregulate meiotic DSB formation as

demonstrated by a dramatic decrease in RAD-51 foci accumulation in aged mated fog-2 mutants, which

is deficient in spermatogenesis and result in a sperm-depleted phenotype after mating, compared to young

and aged unmated fog-2 mutants (Toraason et al., 2022). Though the exact molecular mechanism requires

further inquiry, proteins in the DSB induction process can likely sense signals of sperm and alter their

activity to affect DSB levels in the early meiotic stages. Moreover, mating and compensation of sperm

effects can rescue levels of RAD-51 in aged worms, which may serve as a potential solution to DSB

induction defects (Toraason et al., 2022).

Conclusion

Studying DSB formation and repair, key processes in meiosis, in the C.elegans model reveals the

complicated interplay between individual proteins and structural complexes to regulate the on and off of

DSB machinery. Due to the importance of meiosis in reproduction and development, further research

unveiling the molecular mechanisms of DSB-inducing proteins and how repair is regulated by different

factors such as synaptonemal complexes, cohesins, condensins, as well as age and sperm signals to ensure
appropriate meiotic recombination are needed to deepen people’s understanding of genome stability and

shed new light on this field.

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