Loss of Blm helicase alters circadian period in Drosophila melanogaster

Connor Alexander, Abigail Brown, Ava Hasenoehrl, Jessica Riggs, Eric Stoffregen

ABSTRACT

Bloom syndrome, caused by mutations in the BLM DNA helicase, is characterized by genomic instability and sensitivity to replication stress. Molecular links between DNA damage response pathways and circadian clock function have been identified in multiple organisms — most notably, the clock protein Timeless participates directly in ATR-Chk1 checkpoint signaling, and DNA damage can reset circadian phase by modifying core clock components (Gaddameedhi et al., 2012; Oklejewicz et al., 2008). Our laboratory has previously observed that progeny of Drosophila Blm mutant mothers, which lack maternally-derived Blm during early embryonic divisions, exhibit sleep and circadian abnormalities. Here, we investigate whether Blm deficiency and induced DNA damage affect circadian and sleep behavior in adult Drosophila, using Blm null mutants and heterozygous controls monitored with the Drosophila Activity Monitor (DAM2) system.

Hydroxyurea treatment during development caused differential survival, with Blm mutants surviving at approximately two-thirds the rate of heterozygotes, confirming that treatment caused Blm-relevant DNA damage rather than solely generic toxicity. However, this developmental DNA damage did not translate to circadian dysfunction in surviving adults — no genotype × treatment interactions were detected for any behavioral metric. In contrast, Blm genotype itself had pronounced effects: under constant darkness, Blm mutants displayed significantly higher locomotor activity (p < 0.0001) and a longer free-running circadian period (~23.6 h vs. ~23.2 h; p < 0.001 for all genotype contrasts) independent of treatment.

These findings display a connection between the DNA repair helicase and circadian behavior in Drosophila, establishing further that Blm plays an intrinsic role in circadian period determination distinct from its function in replication stress response.

FIGURES

Fig 1. Blm mutants have longer circadian periods, but hydroxyurea treatment has no period effect.

Box and whisker plots show circadian periods in constant darkness for Blm- and Blm+ (heterozygous control) flies, with and without 30 mM hydroxyurea (HU), measured by DAM2 and analyzed by consensus chi-square / Lomb-Scargle periodogram. The plot shows only rhythmic flies both females (A) and males (B). Boxes show the median and interquartile range; whiskers extend to 1.5 × IQR; the diamond marks the mean; points are individual flies (n below each condition). Blm- flies have a longer period than Blm+ in both sexes (two-way mixed-model ANOVA, genotype p < 0.0001 females, p = 0.0002 males), with no effect of HU (p = 0.17 / 0.70) and no genotype × HU interaction (p = 0.88 / 0.81). The period lengthening is markedly larger in females (~1–1.5 h vs ~0.3 h).

Fig 2. Blm mutant females sleep less during light periods

Total light-phase (daytime) sleep (min) for female Blm- and Blm+ flies ± 30 mM HU, scored at three sleep-bout consolidation thresholds: (A) ≥5 min, (B) ≥30 min, (C) ≥60 min. Boxes show the median and interquartile range; whiskers extend to 1.5 × IQR; the diamond marks the mean; points are individual flies (n below each condition). Blm+ (het) females sleep more than Blm- at every threshold (two-way ANOVA genotype p = 3.3 × 10⁻⁴ / 5.2 × 10⁻⁶ / 2.0 × 10⁻⁵), with the effect strengthening for longer bouts. Female daily activity does not differ by genotype. Blm loss reduces consolidated daytime sleep in females independently of locomotor activity.

Fig 3. BIm mutant males are more active

Mean daily locomotor activity (DAM2 beam crosses) for male Blm- and Blm+ flies ± 30 mM HU, in (A) LD (12:12 light:dark cycles) and (B) DD (constant darkness). Boxes show the median and interquartile range; whiskers extend to 1.5 × IQR; the diamond marks the mean; points are individual flies (n below each condition). Blm- males are more active than Blm+ in both regimes (two-way ANOVA, genotype p = 9.2 × 10⁻⁶ LD, p = 3.2 × 10⁻⁷ DD; no main effect of HU). The genotype effect is robust to the right-skewed Blm+ distribution (Mann–Whitney p = 1.4 × 10⁻⁷ LD, p = 5.7 × 10⁻⁷ DD). Blm loss drives a robust, HU-independent increase in male locomotor activity that reflects a coupled rise in arousal (activity up, sleep down).

Fig 4. HU-induced replication stress selectively reduces Blm mutant survival

Genotype composition of adults eclosing from crosses reared on 0 vs 30 mM HU food, pooled across two trials. (A) Stacked composition of eclosed adults by genotype class (Blm⁻ᐟ⁻ mutant, Blm⁺ᐟ⁻ het, double balancer), labeled with each class's percentage. (B) Ratio of Blm⁻ᐟ⁻ to Blm⁺ᐟ⁻ adults vs HU dose. HU lowered the mutant fraction (18% → 11%) and the mutant:het ratio (0.33 → 0.21), while genotypes carrying a wild-type Blm allele were relatively enriched. Values are descriptive summaries of pooled counts. Developmental HU imposes a genotype-specific survival cost on Blm nulls, confirming that the treatment induces Blm-relevant replication stress in vivo.

CONCLUSIONS

• Loss of Blm function lengthens circadian period

• Loss of Blm function alters sleep/activity

• Hydroxyurea has no robust effect on period, sleep, or activity

• Larval HU selectively kills Blm mutants but does not alter the circadian phenotype

• The Blm circadian phenotype is driven by genotype, not by replication stress imposed during larval development

Acknowledgements:

Project and travel was supported by a SuRE-FIRST award from NIGMS (NIH) #1R16GM146606, an Institutional Development Award (IDeA) from NIGMS (NIH) Grant #P20GM103408, and the Idaho Higher Education Research Council (HERC). Research reported is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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